Sustainable and green natural lignin raw materials are considered as reliable precursors for large-scale production of carbon quantum dots (CQDs). However, extracting CQDs with ultra-low cytotoxicity and favorable fluorescence properties from lignin remains a challenge. Herein, the bio-waste lignin was converted into crude CQDs by an eco-friendly and rapid microwave pyrolysis approach in just 15 min. The as-obtained CQDs modified with nitrogen-atoms (N-CQDs) display a representative graphite-like structure, strong hydrophilicity, tiny particle size of 6.9 nm, and bright green fluorescence with a quantum yield of approximately 22.2% and fluorescence lifetime of 4.29 ns. Moreover, the N-CQDs exhibit high fluorescence stability under the conditions of a range of pH (5–11), continuous UV irradiation (~2 h), and NaCl presence with concentrations of 0–1.2 mol/L. More importantly, the N-CQDs show negligible cytotoxicity. Even at the higher N-CQD concentration (300 μg/ mL), A549 cells still maintain over 90% activity. The N-CQDs could be utilized for fluorescence imaging in living cells based on the above favorable characteristics. The present work paves the way for the value-added utilization of bio-waste lignin and the production of safe cell imaging agents.