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Dissolved oxygen concentration is one of the important indicators of water quality. In order to achieve low-cost and stable measurement of dissolved oxygen in water, this paper reported a single-optical-path dissolved oxygen sensor based upon quenching of single optical path fluorescence. A sol-gel method was used to prepare an oxygen sensitive membrane with tetraethyl orthosilicate (TEOS) and triethoxy(octyl)silane (octyl-triEOS) as the precursor embedded with tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride (Ru(dpp)3Cl2) to optimize the nitrogen-oxygen quenching ratio and improve the performance of the sensor. Optical stability experiments were also conducted. A single-optical-path structure was adopted to reduce complexity, and the fluorescence intensity was calculated using the fast Fourier transform (FFT). The number of calculations were effectively reduced by measuring and storing the fluorescence intensity at 0 mg/L in advance. Calibration experiments were performed on the sensor, and the oxygen sensitive membrane with the highest sensitivity was prepared by the control variable method. The results showed that the fluorescence dyes in the prepared oxygen sensitive membrane were uniformly dispersed, and a fitted Stern–Volmer equation was obtained with a linearity of 0.994 and a sensitivity of 0.115; moreover, the light stability was good. Performance testing and anti-interference experiments were conducted, and the results showed that the standard deviation was 0.076 mg/L and the relative standard deviation was 0.805%. The errors due to changes in salinity and pH were both less than 0.25 mg/L. The sensor offers high accuracy and anti-interference ability, and is suitable for water quality monitoring and marine science. [ABSTRACT FROM AUTHOR]