Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) is a naturally occurring biopolymer belonging to the polyhydroxyalkanoate (PHA) family. Due to its excellent properties (biocompatible, biodegradable, and non-toxic), this biopolymer is presented as a very suitable option for use in regenerative therapy as a drug delivery system (DDS). The protein encapsulated in this study is transforming growth factor β3 (TGF-β3), which plays a key role in the chondrogenic differentiation of mesenchymal stem cells (MSCs). The main objective of this work is to evaluate the efficacy of PHBV nanoparticles (NPs) produced from a dairy by-product (whey) as a DDS of TGF-β3 for cartilage regeneration and extracellular matrix (ECM) synthesis and to reduce the complications associated with multiple high doses of TGF-β3 in its free form. For this purpose, biopolymer cytotoxicity, factor release, cell viability, cell proliferation, and differentiation were analyzed. The results showed that the biomaterial purified with chloroform and ethanol, either by single or double precipitation, was not toxic to cells. A sustained release profile was observed, reaching its maximum around day 4. The TGF-β3 NPs promoted the differentiation of MSCs into chondrocytes and the formation of ECM. In conclusion, PHBV demonstrated its potential as an optimal material for DDSs in cartilage regenerative therapy, effectively addressing the key challenge of the need for a single delivery method to reduce complications associated with multiple high doses of TGF-β3.