Objective To evaluate the clinical effectiveness of venipuncture using the fluorescent intravenous indwelling catheter in dark environment in dog experiments. Methods Six dogs were randomly divided into three groups of two dogs each, and each dog was subjected to 40 times of venipuncture performed on a foreleg with normal intravenous indwelling catheter and fluorescent intravenous indwelling catheter, respectively, under high (101-105 lux), moderate (10.2-100 lux) or low (5-10 lux) brightness conditions. The success rates and time consumption of those two procedures were analyzed and compared statistically. Results The success rate and time consumption for venipuncture using fluorescent and normal intravenous indwelling catheter under low brightness were 82.5% and (204.36±13.13) s vs. 40.0% and (249.35±17.98) s, those performed under moderate (simulating morning and dawn) brightness were 90.0% and (194.86±8.60) s vs. 67.5% and (206.37±9.70) s, all showed a significantly higher efficiency of the venipuncture using fluorescent intravenous indwelling catheter (P < 0.05). Conclusions The use of fluorescent intravenous indwelling catheter has the advantages of higher success rate and shorter time consumption for venipuncture under low and moderate brightness conditions.