In this study, multifunctional fluorescent carbon dots (CDs) were synthesized using a one-pot hydrothermal carbonization reaction, with the naturally-occurring porphyra polysaccharide (PPS) serving as a single carbon source for the first time and ethylenediamine (Ed) acting as the surface passivation agent. The resulting CDs enjoyed a high quantum yield (56.3%), excitation-dependent fluorescence, small size (<10 nm), spherical shape, uniform distribution, positive surface charge, low cytotoxicity and excellent ability to condense macromolecular plasmid DNA. The synthesized CDs were employed for neuronal induction from ectodermal mesenchymal stem cells for the first time via highly efficient non-viral gene delivery. The optimal combination of factors (Ascl1 and Brn2) was selected from seven different combinations out of Ascl1, Brn2 and Sox2 according to the expression of neuronal markers (Tuj1, Map2 and Tau). The results of qRT-PCR demonstrated that the CDs possessed a significantly higher transfection efficiency than the commercially available transfection reagents PEI (25 kDa) and Lipofectamine2000. Moreover, the CDs/pDNA nanoparticles exhibited more efficient neuronal differentiation of the EMSCs than the AT-RA-containing induction medium. Furthermore, the CDs/pDNA nanoparticles could enter cells via both caveolae- and clathrin-mediated endocytosis. Taken together, the natural polysaccharide PPS-derived CDs enriched the current application of CDs by employing the CDs as a novel non-viral gene carrier for neuronal differentiation of adult stem cells, which held great promise in tissue engineering and bioimaging.