Fish behavioral responses are sensitive to chemicals in the water. We tested rare minnow tested for their shoal preference, and the shoal (school) factors including nutritional status, body size, and shoal (school) size that can make their preference most stable were measured. Then shoal preference was measured again while fish and shoal were subjected to a concentration gradient of chemicals (cadmium ion [Cd 2+ ], tricaine methanesulfonate [MS222], and p-chloroaniline). The results showed that single rare minnow preferred shoals over blank control tanks. In addition, this preference was most stable when the shoal was well fed and contained 20 individuals 2 cm long. Although there was no significant response after exposure to p-chloroaniline, the time spent from entering the tank to start moving decreased greatly at concentrations of Cd 2+ >3 mg/L and MS222 >11 mg/L. The time the test fish spent close to the shoal significantly decreased at Cd 2+ >3 mg/L, MS222 >11 mg/L, and p-chloroaniline >10 mg/L, and the frequency of boundary line crossing increased at the same concentrations. The behavioral parameters changed by 20, 5, and 8 min once the lowest-observed-effect concentrations of Cd 2+ , MS222, and p-chloroaniline, respectively, were added. Our study provides useful information on rare minnow shoal preference that may be used for a biological early warning system.