Nanodiamonds were modified such that they carry thiol groups (ND-thiol). Gold nanoparticles were reacted with ND-thiol to obtain a highly stable conjugate of the type ND@AuNPs. Both ND-thiol and the ND@AuNPs were characterized by SEM, TEM, AFM, DLS, zeta potential, XPS, XRD, UV-Vis, Raman, FTIR and cytotoxicity studies. Their biocompatibility was confirmed via an MTT assay with HeLa cells. At a pH value of 6, the ND@AuNPs represent a colorimetric probe that can be used to selectively detect the illegally used β-adrenergic drug clenbuterol (CLB) and the pollutant chromium(III). Detection can be performed visually by monitoring the color change from wine red to purple blue, or by colorimetric measurement of the so-called SPR peaks at 651 and 710 nm. The color changes are due to aggregation, and this is confirmed by TEM and DLS data. The involvement of surface functional groups that assist in analyte recognition was verified by FTIR. The detection limits are 0.49 nM for CLB, and 0.37 nM for Cr(III). The ND@AuNPs were successfully applied to the determination of Cr(III) and CLB in spiked human urine samples. Notably, the low interference by other ions in the detection of Cr(III) in tap and lake water is confirmed by ICP-MS analyses.