Recycling waste biomass into valuable products (e.g., nanomaterials) is of considerable theoretical and practical significance to achieve future sustainable development. Here, we propose a one-pot hydrothermal synthesis route to convert waste tobacco stems into biomass-based N, S-codoped carbon dots (C-dots) with the assistance of carbon black. Unlike most of the previously reported luminescent C-dots, these biomass-based C-dots showed a satisfactory stability, as well as an excitation-independent fluorescence emission at similar to 520 nm. Furthermore, they demonstrated a pH-dependent fluorescence emission ability, offering a scaffold to design pH-responsive assays. Moreover, these as-synthesized biomass-based C-dots exhibited a fluorescence response ability toward tetracycline antibiotics (TCs, e.g., TC, CTC, and OTC) through the inner filter effect (IFE), thereby allowing for the establishment a smart analytical platform to sensitively and selectively monitor residual TCs in real environmental water samples. In this study, we explored the conversion of waste tobacco stems into sustainable biomass-based C-dots to develop simple, efficient, label-free, reliable, low-cost, and eco-friendly analytical platforms for environmental pollution traceability analysis, which might provide a novel insight to resolve the ecological and environmental issues derived from waste tobacco stems.