The detailed flow characteristics in the boundary layer are critical for physical understanding of complex flow and aerodynamic performance optimization of aircraft. In this study, high sensitive micro-electro-mechanical system flexible hot-film sensor was developed for measurement of unsteady boundary layer flow. The effects of annealing temperature and time on the temperature coefficient of resistance (TCR) were studied, the highest TCR value of 5100 ppm °C−1 was obtained after 6 h of vacuum thermal annealing treatment at 400 °C. The flow transition and separation characteristics developed on two-dimensional NACA 0012 airfoil at various Reynolds numbers and angles of attack were investigated by using flexible hot-film sensors with validation by surface pressure distribution and aerodynamic force coefficient. The accurate locations of onset, 50% intermittent and end of transition were detected successfully. The behavior of spatial progression to the leading edge of transition point was found as the Reynolds number and angle of attack increases. The hot-film sensor measurements also demonstrated the static stall of the airfoil was caused by the leading edge separation bubble.