Human pancreatic lipase is a symbolic biomarker for the diagnosis of acute pancreatitis, which has profound significance for clinical detection and disease treatment. Herein, we first demonstrate a paper-based lipase sensor via a phase separation-induced viscosity change. Lipase catalyzes triolein to produce oleic acid and glycerol. Adding an excess of Ca2+ produces calcium oleate. The remaining Ca2+ binds with sodium alginate, triggering hydrogelation with an "egg-box" structure. The viscosity change of the aqueous solution induced by the phase separation process can be quantified by measuring the solution flow distance on a pH test paper. The paper-based lipase sensor has high sensitivity with a detection limit of 0.052 U/mL and also shows excellent specificity. Additionally, it is also utilized for quantitative lipase analysis in human serum samples to exhibit its potency in acute pancreatitis detection. This method overcomes the drawbacks of low sensitivity, slow response, and poor reproducibility caused by the nonuniform distribution of the highly viscous hydrogel on the sensing interface in existing approaches. In conclusion, thanks to the prominent characteristics of high portability, low cost, and easy operation, it is prospective for simple quantitative detection of lipase and has great potential for commercialization.