D-penicillamine (D-PA) plays an important role in medical and clinical treatment of some diseases. In this work, we designed a sensitive fluorometric and colorimetric dual-mode assay on the basis of fluorescent silicon quantum dots (SiQDs) and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) to effectively detect D-PA. The sulfydryl group (-SH) of D-PA can react with DTNB to generate 5-thio-2-nitrobenzoate (TNB), which has a broad absorption peak centered at 407 nm and is capable to absorb excitation light of SiQDs and greatly quench the fluorescence of SiQDs on account of inner filter effect (IFE). Meanwhile, the color of the detection system obviously turned yellow. Consequently, the quantitative determination of D-PA can be achieved through both fluorometric and colorimetric methods. The fluorometric and colorimetric sensing platform can detect D-PA in 1–20 μM and 2–20 μM concentration range, respectively, and the limits of detection (LOD) were 0.48 μM and 0.68 μM, accordingly. Furthermore, the designed sensing platform was utilized to detect D-PA in real biological samples and the experimental results were satisfactory, suggesting the feasibility and potential applications of the sensing platform.