Retinoblastoma (RB) seriously damages children’s vision health and life safety. This study established a new method of optical coherence tomography (OCT) assisted RB zebrafish model construction, and used OCT to evaluate the distribution and survival status of RB cells in the eyes in vivo. Twenty-four normal zebrafish were used in this study, of which 21 were used to construct the RB zebrafish model and the remaining 3 were used as control. The specific method was to inject fluorescent RB cell into the eyeball through an injection under OCT real-time imaging. The distribution and survival status of cell were observed at the 0th, 24th, 48th, and 72th hour after cell injection. At the same time, the zebrafish eyes were imaged with a stereo fluorescent microscope (SFM) before and after modeling, as a control. The results showed that high-resolution, large-depth three-dimensional distribution of RB cell clusters in the eyes could be obtained by OCT. Further OCT image intensity analysis could quantify the number of RB cells, and the results were highly matched with the results of the SFM images. In conclusion, the strategy of combining zebrafish model with OCT imaging technology is expected to provide new methods and equipment for the study of RB.