Stable isotopic content of dissolved organic carbon (δ13C- DOC) provides valuable information on its origin and fate. In an attempt to get additional insights into DOC cycling, we developed a method for δ13C measurement of DOC size classes by coupling high-performance liquid chromatography (HPLC)-size exclusion chromatography (SEC) to online isotope ratio mass spectrometry (IRMS). This represents a significant methodological contribution to DOC research. The interface was evaluated using various organic compounds, thoroughly tested with soil-water from a C3-C4 vegetation change experiment, and also applied to riverine and marine DOC. δ13C analysis of standard compounds resulted in excellent analytical precision (<0.3%). Chromatography resolved soil DOC into 3 fractions: high molecular weight (HMW; 0.4-10 kDa), low molecular weight (LMW; 50-400 Da), and retained (R) fraction. Sample reproducibility for measurement of δ13C-DOC size classes was ±0.25% for HMW fraction, ± 0.54% for LMW fraction, and ±1.3% for R fraction. The greater variance in δ13C values of the latter fractions was due to their lower concentrations. The limit of quantification (SD <0.6%) for each size fraction measured as a peak is 200 ng C (2 mg C/L). δ13C-DOC values obtained in SEC mode correlated significantly with those obtained without column in the μEA mode (p < 0.001, intercept 0.17%), which rules out SEC-associated isotopic effects or DOC loss. In the vegetation change experiment, fractions revealed a clear trend in plant contribution to DOC; those in deeper soils and smaller size fractions had less plant material. It was also demonstrated that the technique can be successfully applied to marine and riverine DOC without further sample pretreatment.