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In this paper, single cell adhesion force was measured using a nanofork. The nanofork was used to pick-up a single cell on a line-patterned substrate inside ESEM. The line-patterned substrate was used to provide small gaps between the single cells and the substrate. Therefore, the nanofork could be inserted through these gaps in order to successfully pick-up a single cell. Adhesion force was measured during the cell pick-up process from the deflection of the cantilever beam. The nanofork was fabricated using focused ion beam (FIB) etching process while the line-patterned substrate was fabricated using nanoimprinting technology. As to investigate the effect of contact area on the strength of the adhesion force, two sizes of gap distance of line-patterned substrate were used, i.e. 1 μm and 2 μm. Results showed that cells attached on the 1 μm gap line-patterned substrate required more force to be released as compared to the cells attached on the 2 μm gap line-patterned substrate.