RP-HPLC coupled with fluorescence detection for separation of carbon nanoparticles (CNP) synthesized with microwave-assisted pyrolysis of citric acid and 1,2-ethylenediamine is presented. The influence of methanol content and pH of mobile phase on the separation of CNP has been investigated. Under optimal mobile phase and elution gradient conditions, the effect of mole ratio of amine to carboxylic groups (NH2/COOH) in the initial reagents on CNP product is studied. At NH2/COOH = 0.67, the strongest fluorescence CNP sample is obtained. The separated CNP fractions are collected and further characterized by UV-visible absorption and photoluminescence (PL) spectroscopy, CE, transmission electron microscopy (TEM), and MALDI-TOF MS. The absorption and PL emission bands of the fractions are bathochromatically shifted with the elution order of CNP on RP-HPLC. The TEM images prove that CNP are eluted from the smallest to the largest. The MS data show that CNP undergo fragmentations, closely relating to their surface-attached carboxylic acid and amide/amine moieties. This work highlights the merit of RP-HPLC coupled with fluorescence detection, TEM, and MS for isolation and characterization of individual CNP species present in a CNP sample.