Purpose: This study was aimed to investigate whether the tumor necrosis induced by radiofrequency ablation (RFA) can improve the ratio of tumor-to-normal tissue (T/NT) after intratumoral injection of 131 I-chTNT. Materials and Method: Eighteen New Zealand rabbits bearing VX 2 tumor on the thigh were randomly divided into two treatment groups (control group: intratumoral injection of 131 I-chTNT alone; RFA group: RFA + intratumoral injection of 131 I-chTNT 3 days after RFA) and each group was further divided into three subgroups I, II, and III (1-2 cm, 2-3 cm, and 3-4 cm in maximum diameter, respectively), by the tumor size. SPECT was performed to evaluate the T/NT on days 1, 8, and 15 after 131 I-chTNT injection. Results: After treatment, all rabbits underwent the SPECT whole-body scan and the T/NT was analyzed. The results showed that T/NT in the RFA group (55.45 -41.83) was significantly higher compared with the control group (7.23 -5.61) (F = 18.89, p = 0.001). Meanwhile, a linear ascending trend was found for T/NT in the RFA group along with the follow-up time (r = 0.47, p = 0.01). The tumor size or the dose of 131 I-TNT injection had no significant effect on the variation of T/NT in both groups ( p > 0.05). Conclusion: RFA before intratumoral injection of 131 I-chTNT can dramatically improve T/NT, demonstrating the potential application of this combination therapy.