A two-fiber probe interrogated by a spectrometer for the measurement of fluorescence emitted from a thin layer of membrane is investigated. For a specific spectrometer, an optimum fiber probe design exists to maximize the sample-probe-spectrometer system performance. In this paper, for the first time, we report that by separating the front end faces of the receiving and illuminating fibers, spectrum resolution and fluorescence collection capability may be simultaneously enhanced. Theoretical and experimental results reveal that such an optimized system collects more emitted rays with incident angles that fall within the full acceptance angle of the slit. The relative collection efficiency increases to 63% when the membrane is positioned very close to the probe tip. By adjusting positions of the receiving fiber and the membrane sample to an optimized combination, we also prove that the optimum performance of spectrometer can be achieved.