In this study, we developed measurement methods of intramolecular carbon isotope ratio of acetaldehyde. First we examined the experimental conditions by an off-line method, which consist of the sealed tube pyrolysis following a gas chromatography / combustion / IRMS (GC/C/IRMS). Next, based on the results of off-line method, we developed an on-line method for a simple, time-saving, high precision (0.5‰ or better) and small sample-size (nanomolar level) procedure measuring the intramolecular carbon isotope ratio of acetaldehyde using gas chromatography / pyrolysis / gas chromatography / combustion / IRMS (GC-P-GC-C-IRMS) coupled with solid-phase microextraction procedure (SPME). This method enabled us to determine stable isotope ratio at the low concentration (10 nmol) with high precision of +0.5‰. This also implied that the on-line method could be applied on determining the intramolecular isotopic ratio of environmental samples. We applied the on-line method on leaves samples.