β-carotene is obtained from both plants and animals and has been the subject of intense research because of its provitamin-A, antioxidant, and anticancer effects. Its limited absorption and oxidative degradation significantly reduce its antitumor efficacy when taken orally. In our study, we utilize a central composite design to develop “bio-safe and highly bio-compatible” solid lipid nanoparticles (SLNs) by using only the combination of palmitic acid and poloxamer-407, a block co-polymer as a surfactant. The current research aim to develop and characterize SLNs loaded with β-carotene to improve their bioavailability and therapeutic efficacy. In addition, the improved cytotoxicity of solid lipid nanoparticles loaded with β-carotene was screened in-vitro in human breast cancer cell lines (MCF-7). The nanoparticles exhibits good stability, as indicated by their mean zeta potential of −26.3 ± 1.3 mV. The particles demonstrated high drug loading and entrapment capabilities. The fabricated nanoparticle's prolonged release potential was shown by the in-vitro release kinetics, which showed a first-order release pattern that adhered to the Higuchi model and showed a slow, linear, and steady release over 48 h. Moreover, a diffusion-type release mechanism was used to liberate β-carotene from the nanoparticles. For six months, the nanoparticles also showed a notable degree of physical stability. Lastly, using the MTT assay, the anti-cancer properties of β-carotene-loaded solid lipid nanoparticles were compared with intact β-carotene on MCF-7 cell lines. The cytotoxicity tests have shown that the encapsulation of β-carotene in the lipid bilayers of the optimized formulation does not interfere with the anti-cancer activity of the drug. When compared to standard β-carotene, β-carotene loaded SLNs showed enhanced anticancer efficacy and it is a plausible therapeutic candidate for enhancing the solubility of water-insoluble and degradation-sensitive biotherapeutics like β-carotene.