Cartilage hypertrophy is a condition in which the cells are completely differentiated, and new morphological changes and mineralization prevent proper cellular functions. The occurrence of hypertrophy during differentiation fails current regenerative strategies for treatment. Strategies to minimize hypertrophy in chondrocytes are categorized into two levels of protein and gene. Among these strategies, one way to affect multiple pathways involved in the development of hypertrophy is to manage microRNA activity in cells. Recent miRNA profiling studies have shown that miR-195-5p upregulates through the transition from chondrogenic to hypertrophic state. Bioinformatics assessment of microRNA targets also indicates that several genes repressed by miR-195-5p play important roles in processes related to hypertrophy. The aim of this study was to develop a microRNA Tough Decoy to suppress miR-195-5p and investigate whether it can prevent a hypertrophic state in chondrocytes. The Tough Decoy (TUD) was designed and evaluated bioinformatically and then cloned into the pLVX-Puro plasmid. The TUD function was validated by Dual-Luciferase assay and qRT-PCR. After delivering TUD to C28/I2 chondrocytes cultured in a hypertrophic medium, hypertrophic differentiation was assessed by histochemical staining, quantitative RT-PCR of hypertrophy marker genes, and alkaline phosphatase activity. Results showed that the TUD could inhibit miRNA efficiently and downregulate hypertrophic markers such as RUNX2, alkaline phosphatase, and collagen 10 significantly compared with the control group. Alcian blue and alizarin red staining also demonstrated the optimal effect of gene constructs on tissue properties and mineralization of the TUD group. Delivering the miR-195-5p Tough Decoy to the cartilage cells can prevent the occurrence of hypertrophy in chondrocytes and could be considered as a candidate for the treatment of other diseases such as osteoarthritis.