Maize (Zea mays L.) performs highly efficient C₄ photosynthesis by dividing photosynthetic metabolism between mesophyll and bundle sheath cells. In vivo physiological measurements are indispensable for C₄ photosynthesis research as photosynthetic activities are easily interrupted by leaf section or cell isolation. Yet, direct in vivo observation regarding bundle sheath cells in the delicate anatomy of the C₄leaf is still challenging. In the current work, we used two-photon fluorescence-lifetime imaging microscopy (two-photon-FLIM) to access the photosynthetic properties of bundle sheath cells on intact maize leaves. The results provide spectroscopic evidence for the diminished total PSII activity in bundle sheath cells at its physiological level and show that the single PSIIs could undergo charge separation as usual. We also report an acetic acid-induced chlorophyll fluorescence quenching on intact maize leaves, which might be a physiological state related to the nonphotochemical quenching mechanism.