Abstract: Two-photon excited fluorescence imaging in the second near-infrared (NIR-II) window has emerged as a promising modality for deep-tissue and high-resolution bioimaging. However, its practical applications are constrained by the insufficient two-photon fluorescence brightness of existing fluorophores. Herein, we investigate the intrinsic mechanism of how structural planar-twisting modulation impacts the two-photon fluorescence brightness of donor-acceptor-donor NIR-II fluorophores. Photophysical characteristics of the chromophores reveal that molecular distortion results in hypsochromic emission and inferior two-photon absorption. Notably, the significantly reduced nonradiative decay rate, primarily attributed to decreased nonadiabatic electronic coupling caused by restricted in-plane vibrations within the molecular backbone, is mainly responsible for the enhanced fluorescence quantum yield and dominates the two-photon fluorescence brightness of the fluorophores. As a result, the moderately twisted chromophore achieves an optimal balance between two-photon absorption and luminescent efficiency on the premise of NIR-II emission, making it a promising candidate as a NIR-II fluorophore. Particularly, there is a strong linear correlation between the electron-hole overlap and two-photon action cross-section of the chromophores, which provides a quantitative indicator for evaluating the two-photon fluorescence brightness of NIR-II fluorophores. These findings highlight the importance of structural modifications in improving the luminescent performance, establishing a theoretical foundation for the rational design of NIR-II chemosensors towards two-photon fluorescence bioimaging applications.