Multifunctional carbon dots have drawn considerable attention due to their potential biomedical application value. We report the preparation of blue-green fluorescence-emitting, multifunctional, nitrogen-and-sulfur co-doped carbon dots (N, S-CDs) synthesized via a one-step process using 1-thioglycerol as a sulfur source, glucose and citric acid as carbon sources, and polyethyleneimine as a nitrogen source. Because of abundant amino and sulfur content, the CDs exhibited high sensibility and selectivity for detecting Cu2+ (detection limit: 0.01 μM, linear range: 0.025 to 50 μM). Fast and sensitive detection of tiopronin was also achieved on the basis of the fluorescence "off-on" mode considering the strong affinity between tiopronin and Cu2+. The N, S-CDs exhibited good biocompatibility as determined by fluorescence imaging using onion epidermal cells and gram-positive bacteria. The CDs also exhibited excellent antimicrobial ability against the gram-positive bacteria. Our results indicate that these novel N, S-CDs could be ideal candidates for several biochemical applications such as antibacterial treatment and detection of small biomolecules.