Objective To study the effects of different color light stimulus, duration of stimulus and day/ night training on the incentive conditioned reflex of rats, and to provide a data basis for the methodology of incentive conditional reflection experiments. Methods Three experiments were included in this study. During the experiment 1, a total of 30 male 8?week?old Wistar rats were divided into three groups: red light stimulation group, yellow light stimulation group and blue light stimulation group. During the experiment 2, a total of 32 male 8?week?old Wistar rats were divided into three groups: the group I (with a light stimulation duration of 10 s), the group II (with a light stimulation duration of 30 s), and the group III (with a light stimulation duration of 60 s). During the experiment 3, a total of 16 male 8?week?old Wistar rats were randomly divided into two groups, of which the first group was trained at 9:00 a. m. every day, and the second group was trained at 21:00 every day. All the laboratory animals were given reward conditioned reflex training, and indicators such as the number of correct nose pokes, the number of incorrect nose pokes, the ratio of correct nose pokes and so on were counted and calculated. Results During the experiment 1, there was no significant difference in the numbers of correct nose pokes and incorrect nose pokes, movement distances and average speeds of the rats among the three groups. During the experiment 2, compared with the group with a light duration of 10 s, the rats in the groups with a light duration of 30 s or 60 s had a higher number of correct nose pokes ( P < 0.05). Moreover, on the 13th and 14th days, the ratio of correct nose pokes of the group with a light duration of 30 s was significantly higher than that of the group with a light duration of 10 s ( P < 0.001). During the experiment 3, there were no significant differences in the number of correct nose pokes and the nose poke accuracy of the rats between the two groups. Conclusions In the reward conditioned reflex test, red, yellow and blue light stimuli have similar reward prediction effect. When stimulated with the same light, the time of stimulation has an impact on the learning ability of the rats, and an appropriate extension of stimulation is beneficial to the formation of conditioned reflex in them. The day/ night training has no effect on the learning and memory ability of the rats.