We report an effective and simple method for separation of carbon nanodots (CNDs) by size-exclusion chromatography that yields CNDs fractions with different sizes and luminescent properties. As a result, three major types of CNDs with spectrally distinct luminescent centers were identified. The first type of luminescent particles has emission maxima at about 400–440 nm, the highest size and luminescence intensity. CNDs of the second type have a middle size and the lowest luminescence intensity with emission maxima in the range 450–470 nm. The distinctive feature of these two fractions is the excitation-dependent shift of the emission maxima to longer wavelengths. The third type of CNDs has luminescence around 520 nm with no emission shift upon the increase of excitation wavelength and the smallest size. The observed spectral variations in different fractions correlate with a change in average particle size as seen in TEM images. Size fractionation of CNDs mixture is very important to many fields, mostly for bioimaging and medicine applications because it leads to CNDs' unified distribution and behavior. Dilution of CNDs in water suspension leads to the increase of emission intensity and shifts the PL spectra to shorter wavelengths providing evidence for concentration quenching effect in CNDs.