Polycystic ovary syndrome (PCOS) is a prevalent and complex hormone-metabolic condition that impacts females of reproductive age. Current pharmacological treatments often address limited symptoms, may involve long-term use of synthetic drugs, and are frequently associated with adverse effects and limited patient adherence. In recent years, natural therapeutic agents such as Lutein and Myo-inositol have acquired increasing interest due to their promising biological properties and minimal side effects. Strong antioxidant lutein aids in lowering inflammation and oxidative stress, while Myo-inositol, a naturally occurring sugar alcohol, has insulin-sensitizing and ovulation-inducing effects, making them ideal candidates for addressing the multifaceted pathophysiology of PCOS. Nevertheless, the application of these compounds in therapy is restricted by their limited solubility, reduced bioavailability, and brief half-life. To overcome these challenges, the present research focused on the development and assessment of sustained-release poly-herbal microspheres incorporating Lutein and Myo-inositol using the emulsification method. Nine different formulations (F1 to F9) were prepared and characterized for in vitro parameters. Among the nine formulations developed, one optimized formulation demonstrated high encapsulation efficiency (>90%), satisfactory production yield (>85%), and sustained drug release of approximately 90% over 8 h. In the letrozole-induced PCOS rat model, treatment significantly reduced malondialdehyde (MDA) levels and increased glutathione (GSH) levels compared with the disease control group (p < 0.001). Histopathological evaluation showed a marked reduction in cystic follicles and restoration of normal ovarian morphology in the treated group. Furthermore, in vivo efficacy was confirmed through histopathological examination of ovarian tissues in animal models, where treated groups, especially T3, receiving the highest concentration demonstrated restoration of normal ovarian architecture, reduction in cystic follicles, and improved follicular development. These outcomes support the hypothesis that a combination of Lutein and Myo-inositol, when delivered through a sustained-release microsphere system, can effectively target oxidative stress, insulin resistance, hormonal imbalance, and ovarian dysfunction, which are central to PCOS pathogenesis.