Carbon dots, which are less than 10 nm in diameter, have been widely investigated because of their unique luminescence properties and potential for use in bioimaging. In the present work, natural carbon dots (L-CDs) were obtained by molecular aggregation, using ethanol-extracted cellulolytic enzyme lignin. The whole process for the preparation of L-CDs was green and simple to operate and did not use toxic chemical reagents or harsh conditions. The newly prepared L-CDs emitted multicolor photoluminescence following one- and two-photon excitation. The L-CDs also showed good cellular biocompatibility, which is crucial for biological applications. One- and two-photon cell-imaging studies demonstrated the potential of L-CDs for bioimaging.