Mesophyll conductance (gm) refers to the diffusion capacity of CO2 inside mesophyll cells, which is the reciprocal of resistance of mesophyll cells. In the early stage of photosynthesis research, mesophyll diffusion resistance to CO2 was usually assumed to be zero, namely the gm was infinite. In recent studies, however, the gm was found to be limited and changed with external environments. As gm directly determines CO2 diffusion and affects leaf photosynthetic efficiency, it is of great significance to mechanestic research of photosynthesis. Presently, simultaneous chlorophyll fluorescence and gas exchange, the curve-fitting and instantaneous carbon isotope (13CO2) discrimination are commonly used to estimate gm, but few literature have been introduced on those methods in China. Therefore, it is particularly necessary to elaborate the principles and processes of these methods and to compare their advantages and disadvantages. We synthesized the relevant literature, and introduced the three methods in detail from the aspects of principle, derivation process and advantages and disadvantages, aiming to provide a methodological basis to promote the research on gm in China. The curve-fitting method was easy to understand and operate. Its fitting model varied with the status of photosynthesis, which is needed to be divided strictly by researchers. Consequently, it was not conducive to be widely used. Although the instantaneous carbon isotope (13CO2) discrimination method improved the accuracy of results, it was complex in measurement and strict in operation. Furthermore, it was less sensitive to test errors with low reliability. Compared with the above two methods, simultaneous chlorophyll fluorescence and gas exchange was more operable and reliable, and was more conducive to the observation and analysis for large samples with multi-processing and multi-repetition. In addition, the use of chlorophyll fluorescence technology not only simplified the test procedures, but also reduced the accidental errors, making the results more scientific. Chlorophyll fluorescence technology also provided saturated pulse activation energy to maximize leaf photosynthetic potential. But this method also had many problems, for instance, to improve the accuracy of chlorophyll fluorescence parameters, a lower gas flow rate was needed, which would increase the risk of gas diffusion and leakage. Thus, this method had a high requirement for a reasonable gas flow rate. In general, simultaneous chlorophyll fluorescence and gas exchange method was most widely used in the actual determination of plant gm.