An optical flow-through biosensor based on the transient retention of p-nitrophenol on an ion-exchange support packed in the flow cell is presented. The approach was applied to the determination of the activity of some enzymes which catalyze the conversion of p-nitrophenyl-derivatives into p-nitrophenol. The method was first developed for determination of p-nitrophenol yielding a linear range between 0.1–5 μg/mL (r2= 0.9922, r.s.d.% less than 2.5), then, it was also applied to the determination of β-D-glucuronidase activity in serum, with a linear range between 0.1–20 U/L (r2= 0.9976, r.s.d.% less than 3.0), and a sampling frequency of 20 h−1. The application of the method to the determination of the enzyme activity in serum samples provided results consistent with those obtained by a conventional method and recoveries within 95–104%.