Spectrophotometry is a simple hemolytic evaluation method commonly used in new drugs, biomedical materials and blood products. It is for the quantitative analysis of the characteristic absorption peaks of hemoglobin. Therefore, it is essential to select the correct detection wavelength when the evaluation system has influences on the conformation of hemoglobin. Based on the study of changes in the characteristic peaks over time of the hemolysis supernatant in four systems, namely, cell culture medium, phosphate buffered saline (PBS), physiological saline and banked blood preservation solution, using continuous wavelength scanning, the selections of detection wavelength were proposed as follows. In the cell culture medium system, the wavelength of 415 nm should be selected within 4 h; , near 408 nm should be selected within 4~72 h. In PBS system, within 4 h, 541 nm, 577 nm or 415 nm should be selected; 4~72 h, 541 nm, 577 nm or near 406 nm should be selected. In physiological saline system, within 4 h, 414 nm should be selected; 4~72 h, near 405 nm should be selected; within 12 h, 541 nm or 577 nm could also be selected. In banked blood preservation solution system, within 72 h, 415 nm, 540 nm or 576 nm should be selected.