Purpose <p>Lymphocyte-activation gene 3 (LAG-3), a next-generation immune checkpoint, has emerged as a promising therapeutic target, but noninvasive tools for evaluating LAG-3 expression remain limited. Herein, we explored an antibody-dependent molecular imaging strategy for noninvasive detection based on a LAG-3-specific antibody, HuL13.</p> Procedures <p>The anti-LAG-3 antibody HuL13 was radiolabeled with ¹²⁴I via electrophilic substitution. LAG-3-expressing A549 cells were constructed by infection with the lentivirus. The specificity and affinity of ¹²⁴I-HuL13 to LAG-3 receptor were evaluated by cell uptake assay and saturation binding assay. Micro-PET/CT imaging studies were conducted in BALB/c nude mice bearing LAG-3⁺ A549 tumors. Immunohistochemistry (IHC) validated LAG-3 expression in tumors.</p> Results <p>The ¹²⁴I-HuL13 exhibited a good radiochemical yield of 95.59 ± 1.27%, high radiochemical purity (RCP, &gt; 99%), and excellent stability. Cell binding assays demonstrated that ¹²⁴I-HuL13 had a higher binding ability to LAG-3⁺ A549 cells compared to control cells. The equilibrium dissociation constant (<i>Kd</i>) of ¹²⁴I-HuL13 was 23.02&#xa0;nM for LAG-3⁺ A549 cells. <i>In vivo</i> pharmacokinetics revealed favorable metabolic stability (t_1/2β = 12.07&#xa0;h). Micro-PET/CT images showed that ¹²⁴I-HuL13 significantly accumulated in LAG-3⁺ A549 tumor from 24&#xa0;h after injection (SUVmax = 0.34 ± 0.03 at 24&#xa0;h), and high contrast tumor to background imaging was clearly observed. IHC confirmed LAG-3 expression correlated with probe uptake.</p> Conclusions <p>¹²⁴I-HuL13 is a novel LAG-3-targeted PET imaging radiotracer with excellent stability. This study highlights&#xa0;¹²⁴I-HuL13&#xa0;as a robust tool for noninvasive LAG-3 imaging, offering potential for optimizing LAG-3-targeted immunotherapy in clinical settings.</p>