We developed a novel single cell measurement of photosynthesis activity using fluorescent oxygen sensor in a microchamber with gas barrier layer. We used Synechocystis sp. PCC 6803, which is a series of cyanobacteria. The chamber wall contains oxygen sensor and gas barrier layer to measure the dissolved oxygen concentration in the chamber. The oxygen concentration is changed by aerobic respiration and photosynthesis of Synechocystis. The variation of the oxygen concentration was used for evaluation of aerobic respiration and photosynthesis of single Synechocystis. To achieve precise oxygen measurement, we used our proposed compensation of photo-degradation of fluorescence. We demonstrated photosynthesis evaluation of single Synechocystis and calculated the photosynthetic performance of sugar production of single Synechocystis.