A straightforward, rapid, and selective fluorescent probe for determination of tilmicosin has been developed based on novel nitrogen and sulfur co-doped CDs (NS-CD). The NS-CDs were synthesized, for the first time, through green, simple one step microwave pyrolysis in only 90 s using glucose as carbon source and l-cysteine as nitrogen and sulfur source. This proposed synthesis method was energy-efficient and resulted in NS-CDs with high production yield (54.27 wt%) and narrow particle size distribution. Greenness of NS-CDs synthesis method was assessed using EcoScale and was proven to be excellent green synthesis. The produced NS-CDs were applied as a nano-probe for determination of tilmicosin in its marketed formulation and milk based on dynamic quenching mechanism. The developed probe showed a good performance for tilmicosin detection in marketed oral solution and pasteurized milk and linearity range of 9–180 μM and 9–120 μM, respectively.