This paper reports a miniaturized fluorescence detection chip with photodiode for quenching assay of atrazine, which effectively reduces the size of fluorescence detection system. The PIN photodiode with fluorescence filter was bonded with PDMS (polydimethylsiloxane) microfluidic chip and was placed just below the microfluidic channel. This detection chip has simple structure consisting of a microfluidic channel and a photodiode. The miniaturized fluorescence detection developed could detect 20nM and it successfully detected the quenching of agricultural herbicide atrazine. The assay result was comparable to that of photomultiplier tube with a fluorescence microscope. This implies the miniaturized fluorescence detection system is applicable to the hand-held diagnostic device or portable environment monitoring system.