Carbon dots (CDs) have recently emerged in biomedical and agricultural domains, mainly for their probe applications in developing efficient sensors. However, the existing high-temperature approaches limit the industrial level scaling up to further translate them into different products by mass-scale fabrication of CDs. To address this, we have attempted to lower the synthesis temperature to 140 °C and synthesized different CDs using different organic acids and their combinations in a one-step approach (quantum yield 3.6% to 16.5%; average size 3 to 5 nm). Further, sensing applications of CDs have been explored in three different biological models, mainly Danio rerio (zebrafish) embryos, bacterial strains, and the Lactuca sativa (lettuce) plant. The 72 h exposure of D. rerio embryos to 0.5 and 1 mg/mL concentrations of CDs exhibited significant uptake without mortality, a 100% hatching rate, and nonsignificant alterations in heart rate. Bacterial bioimaging experiments revealed CD compatibility with Gram-positive (Bacillus subtilis) and Gram-negative (Serratia marcescens) strains without bactericidal effects. Furthermore, CDs demonstrated effective conduction and fluorescence within the vascular system of lettuce plants, indicating their potential as in vivo probes for plant tissues. The single-step low-temperature CD synthesis approach with efficient structural and optical properties enables the process as industrially viable to up-scale the technology readiness level. The bioimaging of CDs in different biological models indicates the possibility of developing a CD probe for diverse biosensing roles in diseases, metabolism, microbial contamination sensing, and more.