The development of enzyme activity analysis methods is critical for precise and rapid assessments of enzyme activity levels within biological systems, facilitating a more profound comprehension of physiological functions and disease mechanisms. Alkaline phosphatase (ALP) participates in various physiological processes involving phosphate ester hydrolysis. Altered ALP activity levels are often indicative of different diseases, underscoring the necessity for accurate ALP activity determination in medical diagnostics. This study innovatively applies turbidity as a physical variable, proposing a turbidimetric sensor based on an enhanced ammonium molybdate reagent for phosphate analysis. By integrating this with the ALP substrate p-nitrophenyl phosphate, a turbidimetric sensor was devised and employed for ALP activity analysis. The proposed turbidimetric sensor demonstrated high sensitivity both for phosphate (0.18 μmol/L) and ALP activity (0.03 mU/mL) assay. In practical applications, this turbidimetric sensor has been effectively employed to detect ALP activity in mouse feces, showcasing its potential for auxiliary diagnosis of inflammatory bowel disease. Significantly, this novel turbidity-based approach offers not only swift and straightforward procedures but also remarkable portability and cost-efficiency. Requiring solely a handheld turbidimeter and eliminating the need for bulky instruments, this approach holds significant potential for point-of-care testing applications.