In present study, heterogeneous Nano-CaO catalyst was synthesized from waste egg shell (WES) and its potential was investigated for direct transesterification of Chlorella pyrenoidosa. Catalyst was synthesized by calcination-hydration–dehydration (C–H-D) process and found to have crystalline structure with average size of 23.65 nm and 64.51 m²/g of surface area as confirmed by XRD and BET analysis. SEM-EDX analysis further confirmed that obtained Nano catalyst is mesoporous with average pore size of 9.28 nm. Response surface methodology (RSM) was applied for optimization of direct transesterification by varying the catalyst dosage (%), reaction temperature (°C) and time (min). The maximum yield of FAME (93.44%) was obtained with combination of 2.06% wt/wt catalyst with 180 min of time and 60 °C temperature. Further, biodiesel was characterized for iodine value, cloud point, pour point, cetane number, higher heating value, acid value and density, which were about 204.14 gI₂/100g,-4.5 °C, 5.4 °C, 32.29, 38.03 MJ/kg, 0.83 mgKOH/g, 0.85 g/cm³ respectively. The reusability and stability of Nano-CaO catalyst was found up to 6 cycles. Findings of this research study support that Nano-CaO catalyst derived from WES is a low cost and sustainable source of catalyst that can be effectively used for biodiesel production.