Mesophyll conductance (gm), the total conductance of CO2 diffusion from substomatal cavity to the site of carboxylation within chloroplast, is a major limiting factor for photosynthesis and a key parameter for improving photosynthetic resource use efficiency of crops. Online 13C discrimination method is an important method for plant eco-physiological studies and a well-established method for measuring gm of C3 plants, although it has not been widely used due to challenges in methodology and high demands on experimental facilities. In this review, we summarized the characteristics of commonly used methods for gm, introduced the basic theory of the online 13C discrimination method, namely Farquhar's photosynthetic 13C discrimination model; systematically introduced the practical measurements, equations and the components of facilities; and reviewed the drivers for variation in gm of C3 plants. At the last part, we discussed the outlook of the development of methodology, new experimental protocols, and applications in measurement scenarios.