Microfluidic chips can be used to realize continuous cryoprotectants (CPA) loading/unloading for oocytes, reducing osmotic damage and chemical toxicity of CPA. In this study, five different Y-shape microfluidic chips were fabricated to realize the continuous CPA loading/unloading. The effects of flow rate, entrance angle, aspect ratio and turning radius of microchannels on the mixing efficiency of microfluidic chips were analyzed quantitatively. The experimental results showed that with the decrease of flow rates, the increase of aspect ratios and the decrease of turning raradius of microchannel, the mixing length decreased and the mixing velocity was promoted, while the entrance angle had little effect on the mixing efficiency. However, the operating conditions and structural parameters of the chips in practical application should be determined based on an overall consideration of CPA loading/unloading time and machining accuracy. These results would provide a reference to the application of microfluidic chip in CPA mixing.