Rapid non-destructive testing of the formation time of blood stains at different dilution ratios under common temperature and humidity conditions was developed using UV visible reflectance spectroscopy. Dilute fresh blood with anticoagulant by 2, 4, 8, 16, and 32 times with tap water. Take 20 μL diluted blood and drop it in the center of a white cotton cloth. After drying, use the reflection accessory of a UV visible spectrophotometer (UV-2450 type) to collect the reflection spectrum, and Excel was used to do regression analysis of trough values and formation time of blood stains at different concentrations. As time increased, there was a significant blue shift in the reflected wave troughs of normal and diluted blood stains near 577.0 nm and 540.0 nm. The values of R² of the regression equation were all greater than 0.800 0, and there was a good linear relationship between the trough blue shift and the formation time of blood stain. The average speed of the trough blue shift increased with the increase of dilution ratio. Conclusion: The blue shift in the reflected wave valleys at 577.0 nm and 540.0 nm can serve as a basis for determining the formation time of normal blood stains, and combined with visual observation, it can also serve as a reference for determining the formation time of diluted blood stains.