Objective To improve fixation methods and tail vein puncture efficiency in conscious rats group．Methods A new four combined holder was designed and made of materials of polystyrene and Polyvinyl chloride.SD rats were randomly divided into A group and B group，twenty rats in each group．Rats in group A fixed by a self-made four combined holder,and rats in group B fixed by common fixture made of organic glass which was commercially available．Then the tail vein injection experiment was conducted respectively by one people．Results It took (9.25士3.70) seconds in group A and （9.95士4.38）seconds in group B to finish the experiment from puncture to see blood return in syringe，and took（47.05士4.65） seconds in group A and （122.45士7.71）seconds in group B to finish the experiment from capture to finish the injection. Futhermore,it was observed （1.4士0.8）times in group A and （2.5士1.2）times in group B with agitation.Conclusion The new four combined rat holder has simple structure, being easy to use, which could significantly shorten the tail vein puncture time and improve the efficiency of the cooperation degree of rats and puncture operation.