Tear glucose is an intriguing biofluid that includes potential biomarkers. While many sensors have emerged nowadays, there is still demand for advanced sensors with nonenzymatic, simple, cost-effective sensing mechanism. Herein, we present a paper-based colorimetric assay by utilizing a gold nanoparticle formation. Experimental characterization substantiated a mechanism in this in situ reduction. Scanning electron microscopy, UV-visible spectrometry, etc. were involved in the scrutiny. As a result, we reached for the rationale whereas the particle formation can be utilized for a glucose sensing using tears. This paper-based detection was insusceptible to physiological tear matrix, i.e., chloride ion effect, false-positive error and synergistic effect by antioxidants. In addition, we evaluated its analytical performance in an artificial tear assay. Of the obtained a linear regressions, the concentration range corresponded to the physiological or pathologic reference range. In addition, within the low-concentration range, a high correlation was resulted 0.965. Furthermore, we investigated statistical validation by employing the Bland–Altman plot. In the end sections of this paper, we denoted its ready-to-use merits by simplicity—as well as the further application of our plasmonic paper.