Resveratrol (3,5,4’-trihydroxystilbene), found in both grape skin and wines, belongs to the poly-phenol group and has been shown to have cancer-preventing properties, boost cardio-protection and antioxidant activity, inhibition of platelet aggregation and anti-inflammatory activity. In this study, a new method for the trace analysis of trans-resveratrol was developed by using solid phase microextraction with on-fiber silylation derivatization. Multidimensional GC equipped with two columns (a non-polar column and a medium polar column) connected in series, a heart-cut valve and cryogenic focusing capacity and coupled with a mass spectrometric detector with simultaneous scan/SIM mode was employed for the chromatographic separation step which increased separation power allows more accurate quantitative results for trans-resveratrol analysis in the complex matrix-wine. The effects of SPME fiber selection, extraction time as well as extraction temperature were investigated. The optimum conditions of derivatization time and temperature were also studied. Calibration curves for peak area were prepared for the different concentration of extracted cis- and trans-resveratrol. The method detection limit of trans-resveratrol based on SPME on fiber derivatization and multidimensional GC-MS with cryotrap and heart-cut was estimated in this study. The content of total resveratrol of six Iowa red wine samples was determined as well.