This paper reports on a high-resolution lensless fluorescence imaging system using membrane deflection for reducing the gap between fluorescent samples and a CMOS image sensor array. The presented system applied the principle of the total internal reflection of excitation light inside a PDMS chip to minimize the exposure of excitation light to the image sensor. We integrated a very thin (5.5 μm) PDMS membrane as a flexible deflector, which allowed for significant reduction of the gap (∼20 μm) inevitably created during the conventional packaging (i.e., wire bonding) of image sensors. In the fabricated system, we achieved minimum distance of 7.5 μm between fluorescent samples and an image sensor array. This reduced gap almost directly corresponds to a resolution as high as ∼7.5 μm, based on the point spread function (PSF).