Preparation of carbon dots (CDs) from biomass waste is of great interest due to its low cost synthesis, environmental compatibility and functionalization without adding dangerous chemicals. Herein, S-doped carbon dot (SCD) was synthesized using agricultural waste as carbon precursors and modified in-situ with rhodamine B dye (SCD@RHB) to construct efficient flouresent probe. SCD@RHB was loaded into HKUST-1 metal-organic framework (SCD@RHB/HKUST-1) and the probe was employed as ratiometric flouresent (RF) sensor for the determination of ciprofloxacin (CIP) antibiotic in trace level. The sensor emits two-resolved peaks at 415 and 575 nm under excitation wavelength of 310 nm which are due to the characteristic fluorescence emissions of SCD and RHB, respectively. Under optimal conditions, the emissions of SCD and RHB change oppositely in the presence of CIP which can be used to evaluate the sensing performance of probe. The ratiometric SCD@RHB/HKUST-1 nanohybrid has a wide linear range (0.1-300 μM) with a low detection limit of 21.3 nM along with good sensitivity and selectivity for CIP. The probe was also used for the determination of CIP in human blood and urine samples. The results demonstrated rapid and accurate analysis with reasonable recoveries of 94 to 102 % for blood sample and 94-106 % for urine sample. This strategy provides a conveninet way to design RF sensors based on HKUST-1.