This dataset contains nifH gene copy numbers of three major marine diazotrophs (i.e. UCYN-A1, Trichodesmium and GammaA) in the surface layer of 519 stations in the North Pacific Ocean. The observation was conducted along 37 trans-Pacific transects between Japan and North America during 2014-2016 as part of the ship-of-opportunity observation operated by the Japanese National Institute for Environmental Studies (NIES). The detailed information of the observation is available at http://nc2.soop.jp/. The nifH gene copy numbers were estimated with quantitative PCR. Additionally, the sampling dates, locations, and physicochemical parameters of each stations are also included in this dataset.