Conventional preparation methods of carbon dots (CDs), generally causing low atom utilization and environmental unfriendliness due to the use of massive solvents, make it difficult for CDs to be synthesized on a large scale. In this work, a one-step solid-phase pyrolysis method was used to address the problems mentioned above. Selecting citric acid and natural biomass glycine as raw materials, through studying influences of the pyrolysis temperature, time, and raw material ratio on properties of the product, it is found that the ratio of citric acid to glycine plays a dominating role to luminescing color and fluorescence intensity of products. Eventually, the glycine-doped blue- and green-emitting fluorescent CDs (GCDs-b and GCDs-g) were prepared by the solid-phase pyrolysis, with high quantum yields of 84.0% and 63.0% and product yields of 71.5% and 64.5%, respectively. Moreover, the uniform and spherical GCDs-b and GCDs-g with average particle size of 4.1 nm and 3.5 nm, respectively, were well-dispersed in aqueous solution, exhibiting excellent and different optical properties, salt tolerance, pH, and light stabilities. Furthermore, in order to explore the influences of surface functional groups on luminescing color of the products, investigation was done on the surface chemical structure, composition, and the characteristics of optical spectra in the formation processes of GCDs-b and GCDs-g. It is rarely reported that the green light emission is related to carbon atom structure and amidation on surfaces of CDs. Additionally, the cellular viability assay of GCDs-b and GCDs-g was also conducted to verify its good biocompatibility, which guarantees these materials can be applied in cellular imaging.