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Using conventional imaging modalities, it is difficult to detect recurrent lesions in prostate cancer patients who have undergone biochemical relapse, especially in patients with low prostate-specific antigen (PSA) levels. We retrospectively reviewed the files of fifty patients with histopathologically confirmed prostate cancer who underwent 99mTc-labeled prostate-specific membrane antigen (PSMA) single-photon emission computed tomography (SPECT)/computed tomography (CT), magnetic resonance imaging (MRI), and bone scan within a 30-day period. PSMA-SPECT/CT indicated metastatic lesions in 39 patients and had a higher detection rate (78.0%) than bone scan (34.0%) or MRI (40.0%). The diagnostic efficiency of PSMA-SPECT/CT imaging for bone and lymph node metastases (50.0% and 42.0%) was better than bone scan (34.0% and 0.0%) or MRI (24.0% and 20.0%). PSMA-SPECT/CT provided a higher detection rate at serum PSA levels of ≤1 ng ml-1, 1-4 ng ml-1, 4-10 ng ml-1, and >10 ng ml-1. No correlation was found between Gleason score, PSA level, and the tracer tumor/background ratio of metastatic lesions. With the aid of PSMA-SPECT/CT imaging, the therapeutic strategy was changed for 31 patients, and this may have enhanced their clinical outcome. In conclusion, PSMA-SPECT/CT imaging could detect more metastatic lesions and achieve a higher detection rate than conventional imaging modalities at different serum PSA levels in prostate cancer patients who had undergone biochemical relapse.