Three-dimensional (3D) in vitro tissue models provide an approach for the systematic, repetitive, and quantitative study of drugs. In this study, we constructed an in vitro 3D acrylated hyaluronic acid (AHA) hydrogel model encapsulating fibroblasts, performed long-period 3D culture, and tested cellular topological changes and proliferation variation in the presence of herpes simplex virus-1 (HSV-1) as an infecting virus and acyclovir (ACV) as the treatment drug. The AHA hydrogels were formed by using Michael addition chemistry of bis-cysteine containing MMP-degradable cross-linker onto AHA prefunctionalized with cell adhesion peptides (RGD). Cellular structures of 3T3 fibroblasts in hydrogel presented different morphological evolution processes and proliferation rates between different groups, including HSV-1 treated alone, ACV treated alone, HSV-1 and ACV cotreated, and control samples. In AHA hydrogel, ACV blocked HSV-1 infection/replication on fibroblasts. Yet, the proliferation of ACV-treated fibroblasts was slower than that of the control group. A significantly longer period was required for cells in 3D AHA gel to regain a healthy status when compared with cells in two-dimensional (2D) culture. This hydrogel-based 3D culture model potentially lays a foundation for analyzing the response of self-organized 3D tissues to viruses and drugs in a way that is closer to nature.