Heart transplantation (HT) is an effective treatment for end-stage heart disease. However, acute rejection (AR) is still the main cause of death within one year after HT. AR is an acute immune response mediated by T lymphocytes, mainly CD4+ T lymphocytes. This study innovatively develops a radiolabeled probe Tc-HYNIC-mAb for noninvasive visualization of CD4+ T lymphocyte infiltration and detection of AR. The Tc-HYNIC-mAb and its isotype control Tc-HYNIC-IgG were successfully prepared and characterized. The specificity and affinity of the probe were assessed by cell-binding experiments. Binding of Tc-HYNIC-mAb to CD4+ T lymphocytes was higher than that of the macrophages and IgG probe groups, and mAb was effective in the blockade of the binding reaction. The biodistribution data confirmed the SPECT/CT images, with significantly higher levels of Tc-HYNIC-mAb observed in allografts compared to allograft treatment (10 mg/kg/d Cyclosporin A subcutaneously for 5 consecutive days after surgery), isografts, or in rats which received allografts injected with Tc-HYNIC-IgG. Histological examination confirmed more CD4+ T lymphocyte infiltration in the allograft hearts than other groups. In summary, Tc-HYNIC-mAb achieved high affinity and specificity of binding to CD4+ T lymphocytes and accumulation in the transplanted heart. Radionuclide molecular imaging with Tc-HYNIC-mAb may be a potential diagnostic method for acute cardiac rejection.