A new two-step calibration method for micro stereovision optical paths is presented and achieved.For one optical path of stereo light microscope（SLM）

the intrinsic parameters can be calculated by the constraint of constant optical operating distance and the magnification obtained from the elementary calibration;Extrinsic parameters can be calibrated from the second step.The average

standard deviation and the relative error of focal length

relative position and magnification of binocular system are gained by the calibration of the SLM under three common-used objective lens magnifications.The accuracy of the calibration is validated by the relative deviation between left and right translation vectors.The calibration method is effective for the micro stereovision system and contributes to an experimental foundation for the analysis of micro channel half-occlusion.