An in-fiber integrated fluorescence online optofluidic sensor was developed by using a hollow optical fiber with a suspended core, in which the micro-region of the sensor for fluorescence quenching reaction was built in the hollow optical fiber. Microholes on the surface of the fiber were etched by a CO2 laser, the fluorescence reagents could be injected into the optical fiber via the microholes and was mixed to form stable microflows in the fiber. Concurrently, indicator molecules were excitated by the evanescent field of the suspended core, the fluorescence was well coupled into the core and was detected in the exited end. The concentration of nitrite was determined by fluorescence quenching reaction during optofluidic reaction in the optical fiber. Experimental results indicate that the microflows could flow through the optical fiber in a short time, therefore, the sensor detects the solution concentration in a fast response rate.Moreover, the fluorescence response of the sensor is nearly linear depending on the concentration of nitrate in the range of 0.1-2.6 mmol/L, which reveals the feasibility of the integrated in-fiber optofluidic method for trace fluorescence detection.