Purpose <p>Given the escalating global burden and high mortality associated with hepatocellular carcinoma (HCC), immune checkpoint inhibitors (ICIs) have emerged as a critical therapeutic approach. T cell immunoglobulin and mucin-domain containing-3 (TIM-3), an emerging immune checkpoint that is highly expressed in HCC, has been linked to poor prognosis due to its association with exhausted T cells and suppressed immune responses. Anti-TIM-3 therapy may hold potential for activating immunity in HCC patients. Stereotactic body radiotherapy (SBRT), a precise radiation technique, can activate the tumor immunity and modulate IC expression. Therefore, the combination of anti-TIM-3 therapy with SBRT is anticipated to enhance the immune response in HCC. An effective measure to evaluate TIM-3 expression after SBRT for improving the synergistic efficacy is needed. This study aimed to develop a non-invasive tool to monitor TIM-3 expression in HCC and optimize the combination of anti-TIM-3 therapy with SBRT to enhance antitumor efficacy.</p> Procedures <p>Clinical data and pathological specimens from HCC patients were collected to evaluate TIM-3 expression in tumor tissues via immunohistochemistry (IHC). A 12-amino-acid peptide targeting TIM-3 was screened via phage display technology and subsequently conjugated with a fluorescent moiety to construct a near-infrared fluorescence (NIRF) probe. The probe’s targeting capability for TIM-3 imaging and its <i>in vivo</i> biodistribution were evaluated using NIRF imaging. After intravenous administration of the TIM-3-targeted probe to mice, dynamic changes in intratumoral TIM-3 expression under varying radiation doses (0/4/6/8&#xa0;Gy × 3F) were visualized via longitudinal optical imaging, identifying the optimal radiation regimen for TIM-3 modulation. Splenic cells were isolated for FCM analysis of TIM-3⁺ cell subpopulations post-SBRT. After determineing the optimal radiotherapy dose, therapeutic efficacy was evaluated in four cohorts: SBRT monotherapy, anti-TIM-3 monotherapy, SBRT-anti-TIM-3 combination therapy, and untreated controls. Tumor regression was monitored via bioluminescence imaging, while splenic CD8⁺ T-cell expansion was quantified by FCM to characterize systemic immune activation.</p> Results <p>TIM-3 was highly expressed in human HCC tissues, with its expression level significantly correlated with tumor stage, vascular invasion status, and patient performance status (PS) score (<i>p</i> &lt; 0.0001). TIM-3 targeting peptides probe was constructed successfully. <i>In vivo</i> fluorescence imaging showed significantly higher tumor-specific fluorescence intensity in mice injected with TIM-3-targeted peptide probe compared to those receiving non-specific probe (<i>p</i> &lt; 0.0001), with peak intratumoral probe accumulation observed at 1&#xa0;h post-injection. Ex vivo organ imaging confirmed predominant probe biodistribution in the liver and kidneys. Radiation dose–response analysis revealed minimal TIM-3 expression in tumors treated with 6&#xa0;Gy × 3F SBRT (<i>p</i> &lt; 0.0001), accompanied by significant downregulation of splenic TIM-3⁺ cells (<i>p</i> &lt; 0.01). Notably, the combination therapy group (SBRT + anti-TIM-3) exhibited superior tumor suppression compared to monotherapy cohorts (<i>p</i> &lt; 0.05), as validated by bioluminescence imaging. Flow cytometry further demonstrated a synergistic increase in splenic CD8⁺ T-cell infiltration within the combination group (<i>p</i> &lt; 0.05), indicating enhanced systemic immune activation.</p> Conclusions <p>This study provides a novel, real-time method to monitor TIM-3 expression in HCC, offering critical insights into SBRT-induced immunomodulation. The combination of SBRT (at the identified optimal dose) with anti-TIM-3 therapy demonstrates enhanced antitumor efficacy, presenting a translational strategy for HCC immunotherapy.</p>