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Efficient and accurate estrus detection is a key management factor determining acceptable reproductive performance in dairy herds. Previous studies have shown that dogs can differentiate between vaginal mucus samples of cows in estrus and vaginal mucus samples of cows in diestrus with an accuracy between 40.3% and 97.0%. We set out (1) to develop a specific training protocol for training dogs to identify cows in estrus from the feed alley and (2) to determine sensitivity and specificity of trained dogs to detect cows in estrus. Six dogs were trained by means of positive reinforcement to detect cows in estrus from the feed alley following the training protocol. Four of those dogs participated in the final test after an average training time of 50 h per dog. Overall, they correctly identified positive cows as being positive in 23 out of 32 cases (i.e. sensitivity of 71.9%) and falsely classified positive cows as being negative in nine cases (28.1% type II errors). Out of 128 cases 119 were correctly classified as true negative cows (i.e. specificity of 93.0%) and in nine cases negative cows were falsely identified as positive cows (7.0% type I errors). Further research is warranted to develop an optimized training protocol that allows training estrus detection dogs for practical use within an appropriate time.