Objective To establish transgenic mouse lineages that can stably express the tandem-dimer tomato (tdTomato) gene and to observe the level of fluorescence expression in tissue sections and stem cells after co-culturing. Methods The gateway clone technique and DNA microinjection were used to construct fertilized ova containing the tdTomato expression vector, which were then transferred to pseudopregnant mice for natural delivery. tdTomato-positive mice were identified and picked using a two-way verification method for breeding and establishment. Neural stem cells from tdTomato-positive mice were co-cultured with bone marrow mesenchymal stem cells and primary neural stem cells from eGFP (green fluorescence protein) mice. Results The tdTomato transgenic mice were successfully constructed, and their lines were established. The biochemical indexes and growth characteristics of the tdTomato transgenic mice did not significantly differ from those of the wild-type mice. Red fluorescence was observed in the tissue sections and brain-derived neural stem cells under fluorescence microscopy. Significant fluorescence was observed at the injection site in the tracer experiments, and two-color fluorescence was stably expressed in the co-culture experiments. Conclusions Stable red fluorescence occurrs in the tissues and cells of the tdTomato transgenic mice, which can be applied to research the direction of differentiation after stem cell co-culturing.