A sol-gel based fiber-optic biosensor with acetylcholinesterase as the biorecognition element has been developed for the rapid determination of organophosphorus pesticides. Nine fluorescent indicators, acridine, acridine orange, neutral red, DAPI, rhodamine B, fluorescein, umbelliferone, FITC on celite and FITC-dextran, have been examined to optimize the fiber-optic system. Results showed that acridine and FITCs were sensitive to the change of pH value caused by the enzyme-substrate catalysis reaction. However, the sensitivity of acridine was 260 times lower than that of FITCs. Higher toxicity of acridine to acetylcholinesterase than FITC was also observed. Moreover, the high-molecular-weight FITC-dextran showed low leakage rate when immobilizing using sol-gel technology, showing that the FITC-dextran was a suitable pH sensitive fluorescent indicator for the OPPs biosensor. The response of the fiber-optic biosensor to the substrate, acetylcholine, was highly reproducible (RSD=3.5%). A good linearity of acetylcholine in the range from 0.5 to 20 mM was also obtained (R2=0.98). Furthermore, a 30% inhibition can be achieved in 30min when 152 ppb paraoxon was added into the system. The results show the possibility for real-time determination of organophosphorus pesticides by using the biosensor developed in this study.