A serum prostate-specific antigen (PSA) measurement method was studied using characteristic spectral imaging method and immunochromatography-based colloidal gold-marked test-strips. An annular light source made of LEDs in the range from 520 to 535 nm irradiated the test-strips sampled by PSA in darkroom, and then the photograph of the test-strip was captured by the CCD industrial camera and transferred to PC. The characteristic value of the test line on the test-strip was acquired by the Quantitative Testing Software on PC using grey level transformation and image segmentation, and then the relationship between the characteristic value and PSA level could be ascertained by cubic polynomial fitting method. Some experiments were completed by 25 blood samples which were taken from one man with no prostate disease and 24 men with prostate disease, and then the correlation coefficient between the measurement values and the true values equals to 0.924. In addition, when the PSA level in the blood sample is lower than 25 ng·mL-1, the measurement accuracy is higher than that when the PSA level in the blood sample exceeds 25 ng·mL-1. The experimental results proved that the proposed method for serum PSA level measurement is feasible.