The effects of temperature and pressure on laser-induced fluorescence (LIF) of OH are numerically studied under the excitation of A–X (1,0) transition at high pressures. A detailed theoretical analysis is carried out to reveal the physical processes of LIF. It is shown that high pressure LIF measurements get greatly complicated by the variations of pressure- and temperature-dependent parameters, such as Boltzmann fraction, absorption lineshape broadening, central-frequency shifting, and collisional quenching. Operations at high pressures require a careful choice of an excitation line, and the Q1(8) line in the A–X (1,0) band of OH is selected due to its minimum temperature dependence through the calculation of Boltzmann fraction. The absorption spectra of OH become much broader as pressure increases, leading to a smaller overlap integral and thus smaller excitation efficiency. The central-frequency shifting cannot be omitted at high pressures, and should be taken into account when setting the excitation frequency. The fluorescence yield is estimated based on the LASKIN calculation. Finally, OH-LIF measurements were conducted on flat stoichiometric CH4/air flames at high pressures. And both the numerical and experimental results illustrate that the pressure dependence of fluorescence yield is dominated, and the fluorescence yield is approximately inversely proportional to pressure. These results illustrate the physical processes of OH-LIF and provide useful guidelines for high-pressure application of OH-LIF.