This study investigated the content of Diazepam(DZP) in feed, nest material, fish bait, water samples and quatic products in aquaculture, and a quantitative detection method using online purification-liquid chromatography tandem mass spectrometry has been established. The results showed that the linearity of DZP content was good in the range of 0.5 to 20μg/L, the correlation coefficient (r2) was greater than 0.999, and the LOQs of this method was 1.0μg/kg, 0.5μg/L, and 1.0μg/kg in fish bait ,water samples and aquatic products respectively. The average recoveries of DZP ranged from 85.2% to 106.5%, 82.3% to 108.0%, and 90.7% to 116.0% spiked at 1.0 μg/kg, 5.0 μg/kg and 10.0 μg/kg with the relative standard deviation (RSD) ranged from 3.6% to 5.1%, 2.7% to 4.3%, 2.1% to 2.6%, respectively. The results showed that DZP was not detected in 7samples of feed for aquaculture,the detection rate of DZP in 28 samples of the nest material and fish bait used in fishing reached 66.7%, with a concentration range from 2.2 to 213090 μg/kg , the detection rate of DZP in 10 samples of the fish attractants reached 100%, with a concentration range from 637 to 3471mg/kg, the detection rate of DZP in 27 water samples was 85.2% , with a concentration range from 0.4 to 9.8μg/L and the detection rate of DZP in 71 fish samples was 54.9%, with a concentration range from 0.5 to 21.8μg/ kg. The use of nest material, fish bait, and fish attractants may be one of the important reasons for the frequent detection of diazepam in aquaculture water environment and aquatic products, which poses potential risks to consumer food safety and the healthy development of the aquaculture industry. It is strengthen the supervision and monitoring of DZP.