The VIS-NIR spectroscopy combined with the improved Moving Window Partial Least-square (MWPLS) method was applied to a high accurate waveband selection for the rapid no-reagent determination of Hemoglobin (HGB) in human whole blood. A new modeling evaluation system was proposed to avoid the evaluation distortion. First, seventy samples were randomly selected from a total of 205 samples as the validation set,the remaining 135 samples were used as the modeling set, and the modeling set was divided into similar calibration (80 samples) and prediction (55 samples) sets for a total of 50 times.Then, modeling and optimization were performed in each division to get stable model. Finally, the optimized model was validated again using the validation set. Experimental results indicate that the VIS-short NIR region 400-1 100 nm can be used as the information waveband of HGB in human whole blood, the global optimal waveband 492-890 nm is further selected from 400-1100 nm with MWPLS method, and a model space including 77 equivalent wavebands is obtained. By taking the 492-890 nm for an example, validation effects V-SEP, V-RP, and V-RSEP are 2.58 g L-1, 0.988, and 1.97%, respectively. It concludes that HGB prediction values of the samples are highly close to the clinic measured values, which may be used in clinical diagnosis.