An aptasensor was designed on a nanocrystalline diamond (NCD) surface that combined with biological recognition elements, PDGF-binding aptamers, which inherently possess high affinity to PDGF-BB proteins. Functional components such as carboxylic acids (-COOH) and amines (-NH2) were directly introduced onto the NCD surface and used as probing units for immobilization of PDGF-binding aptamers. The surface coverage of different components on the NCD was analyzed by X-ray photoelectron spectroscopy (XPS) measurements, and the effects of various functionalizations on the NCD biosensor performance were investigated via fluorescence observations. The coverages of carboxyl and amine groups achieved were 12 and 23%, respectively, for the directly aminated and carboxylated NCD; however, the lower density of carboxyl groups on the functionalized surface did not deteriorate the performance of the COOH-NCD biosensor. Fluorescence investigations demonstrated comparable performance in sensitivity and selectivity for PDGF protein detection on COOH-NCD and NH2-NCD biosensors. Multiple regeneration tests clearly showed that the COOH-NCD biosensor as well as the NH2-NCD biosensor retained a high performance without exhibiting any noticeable degradation.