Purpose <p>A simple and real-time method for the direct and precise identification of safety margins is needed in ultrasound (US)-guided radiofrequency ablation (RFA). Additionally, modulation of the post-ablation immunosuppressive microenvironment is required. The aim of this study was to develop an injectable, ultrasound-visible and immunoregulatory hydrogel for ultrasonic monitoring and adjuvant immune regulation in liver tumor ablation.</p> Methods <p>The experiment was performed in 2 steps. Step 1-The US visibility and stability of the markers were evaluated in vitro in bovine livers, followed by in vivo animal test in beagles. Step 2-Evaluation of the antitumoral effect after RFA of the specific US marker F127-MB@MSA-2, which has immunomodulating properties, in murine in vivo animal tumor models.</p> Results <p>In step 1, 24 candidate materials were tested for ultrasound visualization in bovine liver. Among these, air, 5&#xa0;mm metal stick, Pluronic F127 hydrogel microbubbles (F127-MB), polyglycolic acid (PGA) absorbable sutures, ultrasonic couplant, and calcium alginate demonstrated clear ultrasonic visibility and enduring stability in vitro and in vivo. In step 2, the antitumoral effect of F127-MB@MSA-2 after incomplete RFA was demonstrated in murine H22 and CT26 tumor models. Tumor Flow cytometry and immunofluorescence staining revealed that F127-MB@MSA-2 gel reduced the infiltration of tumor-promoting cells, while it increased the proportion of CD8⁺ T cells within the tumors after RFA.</p> Conclusion <p>Using animal models, we demonstrated the feasibility of US-guided administration of the multifunctional gel (F127-MB@MSA-2) prior to RFA, which helped to accurately ablate tumors and increase the anti-tumor effect.</p> Graphical Abstract <p></p>