• A new deep-learning-based individual stoma tracking pipeline was proposed. • The circadian rhythm of stomata opening was first reported from video data. • Smaller stomata not only respond faster but also had longer closure time at night. Plant stomata are essential channels for gas exchange between plants and the environment. The infrared gas-exchange system has greatly accelerated the studies of stomatal conductance ( g s ). Nevertheless, due to the lack of in-situ monitoring techniques, the behavior of stomata themselves remains poorly understood, especially in nocturnal environmental conditions. Here, a deep-learning-based stoma tracking pipeline ( StomataTracker ) was first proposed to continuously monitor stoma traits from unprecedentedly long-term, continuous, and non-destructive video data. Compared to the semi-automatic method (ImageJ), the open-source StomataTracker could greatly improve the extraction efficiency from 207 s to 1.47 s of stomatal traits, including stomatal area, perimeter, length, and width. The R 2 adjusted of the four stomatal traits ranged from 0.620 to 0.752. In addition, the rhythm of wheat stomata opening in a completely dark environment was first reported from long-term video data. The closed time of stoma at night was negatively correlated with stomatal traits, and the R ranged from −0.583 to −0.855. The heterogeneity of stomatal behavior also highlighted that smaller stomata have the rhythm pattern of longer closure time at night. Overall, our study provides a novel perspective for stomatal study, and it is conducive to accelerating the application of stomatal circadian rhythm in wheat breeding.