Tebuconazole is a chiral trizole fungicide and widely used in many crops for controlling disease. Tebuconazole is potential toxic to some aquatic organisms but relative information of its isomers is scarce. To detect the endocrine disrupting effects and difference of rac-, R-, and S-tebuconazole, the chitinase activity in Daphnia magna and chitobiase activity in each test medium were used as biomonitors after a 14-day exposure. Results showed that chitinase activity was significantly reduced by rac-, R-, and S-tebuconazole. The chitobiase activity in the test medium was reduced by rac- and R-tebuconazole before day 10, and only one peak was observed at day 10 or day 12 compared with two obvious peaks in the control group (days 6 and 12). S-tebuconazole delayed and reduced the reproduction of D. magna, but did not delay the first chitobiase activity peak, whereas the second peak could not be characterized as the exposure concentration and time increased. Compared with chitinase activity, chitobiase activity can still be used as a rudimentary model for identifying molt-interfering xenobiotics, and further studies should focus on the analysis of correlations between these parameters.