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A simple, one-step and facile method has been introduced to prepare fluorescent and electrochemically active carbon nanoparticles with single-size distribution and good long-term stability by electrochemical exfoliation of polyacrylonitrile-based carbon fibers in an alkaline solution-phase condition. The preparation condition was systematically optimized by studying the effect of temperature and electrolytes. It has been found that an electrochemical exfoliation reaction carried out at an applied potential of 2 V vs. Ag/AgCl in a phosphate-ion-containing alkaline solution at a temperature of 40 °C is an ideal condition for the preparation of 14 ± 4 nm-sized carbon nanoparticles. Unlike the literature protocols, there are no filtration and membrane dialysis-based off-line sample pretreatments adopted in this work. The as-prepared carbon nanoparticles were characterized by fluorescence, Raman spectrum, transmission electron microscope, and X-ray photoelectron spectroscopic characterization methods. It was found that the carbon–oxygen functional group rich in graphene–oxide quantum dots (GOQDs) such as carbon nanoparticles were formed in this work. A preliminary study relating to simultaneous electrochemical oxidation and the sensing of uric acid and ascorbic acid with well-resolved peaks was demonstrated as a model system to extend the new carbon material for electroanalytical applications. Furthermore, in situ synthesis of 2 nm-sized gold nanoparticles stabilized by GOQDs was presented. The carbon nanoparticles prepared by the direct method in this work have shown good stability over 6 months when stored at room temperature. The electrochemical exfoliation reaction has been found to be highly reproducible and suitable for bulk synthesis of luminescence-effective carbon nanoparticles to facilitate fundamental studies and practical applications.