To investigate the transition and separation of boundary layer in LP turbine with low Reynolds number conditions, and grope for the application prospect of surface hot-film testing technique in rotating component turbine environment, test research of a certain large-size low-speed two-stage LP turbine was hold. Test method and data processing method were detailed described. Comprehensive analysis of the transition and separation flow on the surface of the second stator in typical low Reynolds number condition was conducted, and the influence of Zweifel coefficient was debate. Result shows that surface hot-film testing technique is an effective method to investigate calmed region phenomenon and unsteady flowing effect in turbine stages. To acquire valid experimental data, the insensitivity of sampling rate and sampling time should be verified by anchoring blade passing frequency and blade passing period respectively. The wake-induced transitional stripes, identified as trajectories of upstream rotor blade, occupy one quarter of periodic passing channel, while the derivative calmed regions restrain natural transition process, conversely. With the decrease of turbine speed, transition point on the suction side of the second stator moves forward along with transition region extended.