Abstract: Poly-L-Lysine (PLL) with a molecular weight of 150000—300000 is often used in cell culture and histology to promote cell or tissue adhesion. After treating the cover glass with 0.05%—0.1% PLL solution, PLL adhered to the surface of the cover glass to form a monolayer. The density of the PLL directly affects the adhesion effect of the cover glass. However, because PLL is colorless and transparent, it is difficult to distinguish the PLL density and distribution on the surface of PLL-treated cover glass with the naked eye, so it is crucial to establish a simple and effective PLL detection method. However, there are still no relevant methods reported worldwide. In this study, a laboratory visual and quantitative detection method of PLL was developed. In this method, the fluorescein isothiocyanate (FITC) is added to the side chain amino group (-NH2) of the PLL on the surface of the cover glass in sodium bicarbonate solution, and fluorescence imaging is applied to detect the FITC-PLL. In this study, we firstly coated cover glass of 0.1% PLL of three different brands (S, M and A), and found that only the brand S PLL-coated cover glass still had intact tissue section after several washes with phosphate buffer. Accordingly, we found that the FITC-PLL of brand S on the cover glass was bright and uniform, while both the FITC-PLL of brand M and A were dim (3.4% for M, and 4.5% for A compared with S). Therefore, this method is suitable for the quality control of the homemade or commercial PLL-coated cover glass. Furthermore, the method also provides a reference for the establishment of a visual and quantitative detection method of material with a -NH2 group attached to glass or plastic surface.