Bright two-photon fluorescent probes are highly desirable to be able to optically probe biological activities deep inside living organisms with larger imaging depth, minor autofluorescence background, and less photodamage. In this study, we report the biocompatible nitrogen-doped graphene quantum dots (N-GQDs) as efficient two-photon fluorescent probes for cellular and deep-tissue imaging. The N-GQD was prepared by a facile solvothermal method using dimethylformamide as a solvent and nitrogen source. The two-photon absorption cross-section of N-GQD reaches 48,000 Göppert-Mayer units, which far surpasses that of the organic dyes and is comparable to that of the high performance semiconductor QDs, achieving the highest value ever reported for carbon-based nanomaterials. More importantly, a study of penetration depth in tissue phantom demonstrates that the N-GQD can achieve a large imaging depth of 1800 μm, significantly extending the fundamental two-photon imaging depth limit. In addition, the N-GQD is nontoxic to living cells and exhibits super photostability under repeated laser irradiation. The high two-photon absorption cross-section, large imaging depth, good biocompatibility, and extraordinary photostability render the N-GQD an attractive alternative probe for efficient two-photon imaging in biological and biomedical applications.