We developed a rapid and sequential analysis system to determine stress marker in the milk. One of the famous stress markers, cortisol, was detected using our method. Quartz crystal microbalance (QCM) method with a twin sensor was used in this study. One channel detected stress marker corresponding to antigen-antibody interaction and the other channel was used as a reference to remove environmental influences. Although nonspecific adsorption was monitored on each channel, frequency difference between them was within a few Hz on the injection of sample solution. One determination cycle including regeneration step could be performed within 10 minutes. The system could detect the cortisol level from 0.1 pg/mL to 100 pg/mL. These results show that our system has a potential to check the daily feeding condition for cows in terms of animal welfare.