A quartz crystal microbalance with dissipation (QCM-D) analysis was used to investigate fetal bovine serum (FBS) protein preadsorption on a hydroxyapatite (HAp) surface and the subsequent adhesion process of fibroblasts as compared with the case of oxidized poly(styrene) (PSox). The results showed that the preadsorption of FBS proteins on HAp promoted the subsequent initial cell adhesion ability. Moreover, the measured frequency (Δf) and dissipation shift (ΔD) curves, ΔD-Δf plots and viscoelastic analysis were used to study the initial cell adhesion process in real time. It was suggested that FBS-HAp showed sensitive changes in mass and viscoelasticity as compared with FBS-PSox, which realized the in situ reflection of the cell adhesion state, and the interfacial reactions between the cells and FBS-HAp surfaces such as dehydration and binding occurred to promote the initial cell adhesion and spreading. The viscoelastic analysis of the interface layer showed that the adhered cells on FBS-HAp could secrete some viscous substances such as extracellular matrix (ECM) proteins at the interfaces to provide good adhesion behaviors, and the Voigt-based viscoelastic model could clearly reveal the cellular interfacial viscoelasticity depending on the substrate surface. In addition, the morphology of cells was observed by confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM), and it was found that the pseudopodia were more uniformly stretched on FBS-HAp than on FBS-PSox. Furthermore, the state of the interfacial protein layer was analyzed by localized Fourier-transform infrared (FT-IR) spectroscopy and fluorescence microscopy (FLM), and it was indicated that the type of substrate affects the formation state of ECM proteins, resulting in changes in cell adhesion properties and morphology. The abundant formation of connective proteins (i.e., collagen type I) on FBS-HAp promoted subsequent pseudopodia formation and cell spreading. Therefore, the initial adhesion properties of fibroblasts on the FBS-HAp surface were systematically studied, which is of great importance for understanding the interfacial interaction between biomaterials and cells, and has great application value in biomedical fields.