Nonylphenol polyethoxylates (NPEOs) is an important kind of nonionic surfactant widely used in the manufacturing and life of human beings. A second derivative ultraviolet spectrum method was established for the determination of the total NPEOs (the average n was 10) in microbial degradation water samples, and the optimal experimental conditions were studied. Water was used as the solvent. The derivative peaks of 283 nm and 288 nm were characteristic peaks, and the vertical distance of their culminations was used as the quantitative basis. Under the optimal experimental conditions, the standard curve of NPEOs was linear within the range of 1.8~440 μg/mL. The linear correlation coefficient was 0.9995, and the detection limit was 0.5 μg/mL. Actual NPEOs degradation water samples were determined. The recoveries of NPEOs ranged from 98.1% to 104.4% and the relative standard deviation varied from 1.9% to 2.1%. The results showed that the second derivative ultraviolet spectrum method was simple and fast, and was suitable for the determination of the total NPEOs in microbial degradation water samples.