<p>The increasing scarcity of petroleum-based bitumen and its adverse environmental and economic implications necessitate the development of sustainable alternatives for flexible pavement construction. Bio-bitumen is an environmentally friendly substitute for conventional bitumen and is renewable and cost-effective. This study explores the potential of castor oil (CO) and rice husk ash (RHA) as bio-based modifiers in the formulation of castor oil and rice husk ash-modified bio-bitumen (CORMBB). The optimization of CO (5–15%) and RHA (2–6%) content was conducted using response surface methodology (RSM) with central composite design (CCD) and a genetic algorithm-optimized artificial neural network (GA-ANN) model. Binder properties, such as penetration and softening point, along with bituminous mixture performance indicators, including Marshall stability, indirect tensile strength (ITS), tensile strength ratio (TSR), and Cantabro loss, were considered response variables. The GA-ANN model exhibited high predictive accuracy, identifying optimal dosages of 9.02% CO and 5.85% RHA, while RSM yielded 8.992% CO and 6% RHA. Subsequently, the optimized CORMBB (9% CO and 6% RHA) was subjected to storage stability test, X-ray Diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and dynamic shear rheometer (DSR) analyses to assess, its compatibility of CORMBB, chemical interaction among CO, RHA and base binder (BB), and rheological behavior. The findings indicate that CO enhances flexibility, adhesion, and moisture resistance, whereas RHA improves stiffness, rutting resistance, and aggregate retention. FTIR and XRD results confirmed improved molecular interactions and structural integrity, underscoring the potential of CORMBB as a sustainable, eco-friendly alternative to conventional bitumen. This study provides comprehensive insights into the formulation and performance of bio-modified binders, promoting the integration of agricultural by-products in pavement applications.</p>