With rapid development of industrial processes, in particular more recently influenced by “Haze”, air quality draws more and more attentions. A refill of oxygen in air is one of crucial solutions to improve air quality. In contrast to conventional technologies for oxygen production (i.e. physical separation of air, chemical reactions, water electrolysis), the innovative technology of electrochemical continuous separation of oxygen from air features separation of pure oxygen from air, high efficiency, continuous operation, environment friendly, silent operation, ease of scale up and applicability to indoor or outdoor fields. This technology involves two crucial components of polymer electrolyte fuel cells and solid polymer electrolyte water electrolysis (abbreviated as fuel cell and electrolyzer). In this article, the effect of operation conditions on single cell performance such as operation temperature, reactant gases utilization ratios, relative humidity and pressure, etc. for fuel cell was investigated, as well as the ways of water supply (at anode and/or cathode), water flow rate and operation temperature, etc. for electrolyzer. In terms of fuel cell, the polarization curve was measured, the electrochemical impedance spectra were conducted and the ionic conductivity and activation energy of Nafion® membrane were calculated. Polarization curve was fitted to obtain intrinsic parameters including Tafel slope, exchange current density of oxygen reduction reaction (i0) and m, n, related to mass transfer etc. It showed that the optimum of fuel cell was under conditions of ambient pressure, 60℃ of operation temperature, 0.42 W·cm-2 of peak power density, 77 mohm·cm2 of cell areal resistance (membrane) and 41.4 mS·cm-1 of ionic conductivity. The Tafel slope slightly varied with temperature, ca. 120 mV·dec-1, but was influenced by the relative humidity. The relative humidity remarkably affected the fuel cell performances. In electrolyzer, the optimum was under conditions of 65℃ of operation temperature, 1.08 ohm·cm2 of cell areal resistance and 11.7 mS·cm-1 of ionic conductivity. The effect of water flow rate on performance was negligible. The ways of water supply follow an order of both anode and cathode≈anode>cathode. Under above conditions, activation energies of Nafion®211 and Nafion®115 membranes were calculated as 3.75 and 4.61 kJ·mol-1, respectively. Based on the optimum of single cell performances of fuel cell and electrolyzer, in this article, the preliminary experimental data were provided for the subsequent implementation of scale up of cell stack system for oxygen production.