A ratiometric optical sensor has been developed with electrospinning processing method for dissolved oxygen measurement. The sensing film is fabricated by using silver nano-particles (Ag NPs) doped with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride complex (Ru(DPP)3Cl2) encapsulated in plasticized polymethyl methacrylate (PMMA). An insensitive 3-(2-benzothiazolyl)-7-(diethy lamino)-(6CI,7CI) (Coumarin6) is adopted as reference. The ratio of oxygenation is calculated at each image pixel of a 3CCD camera to quantify the oxygen concentration in aqueous environment. Compared to Ag-free film, the response time of Ag-containing films were improved from 1.5 s to 1.0 s upon switching from deoxygenated to air saturation and from 65 s to 45 s from air saturation to fully deoxygenated. The response times of the Ag-free film obtained by knifing was 2.0 s upon switching from deoxygenated to air saturation and 104 s from air saturation to fully deoxygenated. Results of the evaluation of accuracy, limit of detection, stability, and photostability are presented. An experiment measuring the spatiotemporal variation of oxygen distribution within the photosynthesis and respiration of Chlorella vulgaris is demonstrated. It is shown that the nanofiber-based optical sensor film could serve as a promising method for rapid oxygen monitoring in aqueous applications.