Objective To study feasibility of retrograde infusion of hyperpolarization-activated cyclic nucleotide-gated channel protein 4 (HCN4) and connexin fluorescent dye (Alexa Fluor 633)-labeled antibodies through the aorta to image the cardiac conduction system (CCS) in rat hearts. The optimal dosage, infusion time and photochemical stability of fluorescent dyes were also studied. Methods Ex vivo rat heart anterograde perfusion models were established in 33 male SPFSD rats, and the primary and secondary antibody solutions were injected sequentially. The atrioventricular junction was observed and the fluorescence intensity of the area was recorded when the perfusion reaches the scheduled time. We set five dose gradients (3 rats per gradient), 5 perfusion time gradients (three rats per gradient) and 10 LED continuous illumination time gradients in 3 rats under specific dose and perfusion time, the fluorescence intensities of the region were observed and recorded. Standard immunofluorescence stained paraffin sections and frozen sections were prepared for histological comparison. Results A HCN4 red fluorescence signal aggregation region was observed in the atrioventricular junction, which was identified as the AVN structure based on HCN4/Cx43 semi-quantitative fluorescence intensity analysis and histological comparison. With increasing antibody perfusion time, both AVN and background fluorescence intensity showed no statistically significant difference. The ratio of AVN to background fluorescence intensity also increased with the increasing antibody perfusion time. When the illumination time of AVN was prolonged, the fluorescence intensity of both AVN and background showed a downward trend but no statistically significant difference. Conclusion The anterograde perfusion with fluorescent dye (Alexa Fluor 633)-labeled antibody can successfully image the AVN of the CCS in ex vivo rat hearts under stereoscopic fluorescence microscopy. Increasing the antibody dose results in different AVN imaging effects. The imaging effect of AVN improves with an increase antibody perfusion time. Even after long-term (8 h) exposure to light, Alexa Fluor 633 can still maintain a certain level of AVN image.