Lipase activity was detected based on the fluorescence properties of gold nanoclusters (AuNCs) and the inner filter effect (IFE) of p-nitrophenol. Glutathione-modified AuNCs was used as fluorophore, and p-nitrophenol produced from the lipase-catalyzed hydrolysis of p-nitrophenol palmitate as fluorescence absorbent. The results showed that under the optimal conditions (p-nitrophenyl palmitate concentration 1.6 mg/mL, pH 7.5, temperature 50 ℃, and reaction time 20 min), the relative fluorescence intensity (F₀–F, y) was positively correlated with lipase activity (x) in the range of 5.6‒196 U/L, which was described by the equation y =2.0035+0.9368x, with a correlation coefficient (r²) of 0.9978, and the detection limit was 1.3 U/L (at a signal-to-noise ratio of 3). The developed method is simple and sensitive, and can be applied to the detection of lipase activity.