In this work, poly(sodium 4-styrenesulfonate) (PSS) modified molybdenum disulfide quantum dots (MoS2-PSS QDs) were synthesized via a simple hydrothermal method using l-cysteine and anhydrous sodium molybdate as precursors and PSS as a modification reagent, and a selective and sensitive fluorescent sensing method for the determination of p-nitrophenol (p-NP) based on their UV emission was developed. The obtained MoS2-PSS QDs have an obvious UV emission peak (390 nm) with quantum yield of 5.13%. The strong absorption peak of p-NP at 400 nm has large spectral overlap with the UV emission peak (390 nm) of MoS2-PSS QDs. Because of this p-NP absorption, the fluorescence of MoS2-PSS QDs at 390 nm is quenched with the introduction of p-NP via the inner filter effect (IFE) and the decreased fluorescence intensity was linearly proportional to the p-NP concentration in the range of 1–20 μmol/L, leading to a detection limit of 0.13 μmol/L for p-NP. The MoS2 QDs-based fluorescent probe for p-NP is sensitive and selective and was successfully applied in the determination of p-NP in the pond water samples with satisfactory results.