Studying the microbial activity of bacteria in water and rapidly assessing the growth status and reproductive capacity of bacteria in water environments are of great significance for the scientific management of water resources and the guarantee of public health security. Although traditional bacterial activity detection techniques, such as the colony counting method, staining observation method and molecular biology methods, are effective, they have cumbersome operation procedures, consume a lot of time and workforce, and are difficult to meet the needs of real-time online detection. In response to the drawbacks of traditional bacterial activity detection techniques, a novel rapid detection method for bacterial activity based on multi-wavelength transmittance spectra is proposed. Taking the common Escherichia coli (E. coli) in water as the research object, the transmittance spectra of E. coli within the wavelength range of 190～800 nm were measured using an ultraviolet-visible spectrophotometer. The characteristics and regularities of the transmittance spectra of E. coli under different proportions of viable bacteria were thoroughly studied. A relationship model between the ratio of double-wavelength optical density and bacterial activity was constructed through the systematic analysis of the correlation and sensitivity of the transmittance spectra of E. coli with changes in activity at different wavelengths. Based on this model, the bacterial activity was calculated, and the accuracy and stability of the calculation results of bacterial activity under different double-wavelength ratio methods were compared and analyzed. The results show that: (1) Due to the differences in the contents of biomolecules in bacteria with different activities, within the wavelength range of 230～300 nm, the bacterial solutions containing higher proportions of viable bacteria have higher optical densities. (2) By analyzing the sensitivity and correlation of the transmittance spectra of E. coli in the wavelength range of 230～300 nm with the activity changes, it was found that the correlation coefficient ranges between the activity of E. coli and the optical densities is 0.959 2～0.993 3, which preliminarily determines the optimal wavelength band for quantitative determination of bacterial activity. (3) Selecting 230 nm as the measurement wavelength and 670 nm as the reference wavelength, a fitting curve of the ratio of optical densities at the two wavelengths to bacterial activity was constructed. The correlation coefficient reached 0.946 2, and the detection range of the viable bacteria proportion was 0%～100%. (4) This fitting curve determined the activities of three E. coli bacterial solutions with different activities. Compared with the plate colony counting method, the maximum relative error was 3.70%, the average relative error was 1.43%, and the accuracy was optimal. This method only requires the optical densities at 230 and 670 nm, and the detection time is within 1 second, which can complete the rapid and accurate detection of bacterial activity. This research achievement provides a new technical idea for the rapid detection and early warning of bacterial activity in water and has potential application value.