Natural biomass is used for facile synthesis of carbon quantum dots (CQDs) with high fluorescence, owing to its abundance, low cost, and eco-friendliness. In this study, a bottom-up hydrothermal method was used to prepare CQDs from water hyacinth (wh) at a constant temperature of 180 °C for 12 h. The synthesized wh-CQDs had uniform size, amorphous graphite structure, high water solubility (containing multiple hydroxyl and carboxyl groups on the surface), excitation light-dependent characteristics, and high photostability. The results showed that the aqueous solution of CQDs could detect Fe³⁺ rapidly, sensitively, and highly selectively with a detection limit of 0.084 μM in the linear range of 0–330 μM, which is much lower than the detection limit of 0.77 μM specified by the World Health Organization. More importantly, because the wh-CQDs were synthesized without any additives, they exhibited low toxicity to Klebsiella sp. cells even at high concentrations. Moreover, wh-CQDs emitted bright blue fluorescence in Klebsiella sp. cells, indicating its strong penetrating ability. Correspondingly, the fluorescent cell sorting results also revealed that the proportion of cell internalization reached 41.78%. In this study, wh-CQDs derived from natural biomass were used as high-performance fluorescent probes for Fe³⁺ detection and Klebsiella sp. imaging. This study is expected to have great significance for the application of biomass carbon spots in the field of cellular imaging and biology.