Antibiotics are commonly used as antibacterial medications due to their extensive and potent therapeutic properties. However, the overconsumption of these chemicals leads to their accumulation in the human body via the food chain, amplifying drug resistance and compromising immunity, thus presenting a significant hazard to human health. Antibiotics are classified as organic pollutants. Therefore, it is crucial to conduct research on precise methodologies for detecting antibiotics in many substances, including food, pharmaceutical waste, and biological samples like serum and urine. The methodology described in this research paper introduces an innovative technique for producing nanoparticles using silica as the shell material, iron oxide as the core material, and carbon as the shell dopant. By integrating a carbon-doped silica shell, this substance acquires exceptional fluorescence characteristics and a substantial quantum yield value of 80%. By capitalising on this characteristic of the substance, we have effectively constructed a fluorescent sensor that enables accurate ofloxacin analysis, with a detection limit of 1.3 × 10⁻⁶ M and a linear range of concentrations from 0 to 120 × 10⁻⁶ M. We also evaluated the potential of CSIONPs for OLF detection in blood serum and tap water analysis. The obtained relative standard deviation values were below 3.5%. The percentage of ofloxacin recovery from blood serum ranged from 95.52% to 103.28%, and from 89.9% to 96.0% from tap water.