Escalating environmental concerns associated with the usage of plastic leads to the present study for developing a novel natural based biodegradable starch film with nanocellulose and nanohemicellulose reinforcements. An effective sequential strategy for enhancing the yield of high purity cellulose and hemicellulose from rice straw was developed by maximum lignin removal through microwave pre-treatment and complete separation of cellulose and hemicellulose by combined ultrasonic-alkali treatment. The two-step approach of microwave-assisted delignification and ultrasonic extraction produced a delignification of 84.51 ± 1.21% and a yield of 0.282 ± 0.011 g/g cellulose and 0.19 ± 0.008 g/g hemicellulose. The proposed methodology produced cellulose of purity 93.37 ± 2.43% and hemicellulose of purity 98.6 ± 1.32%. The cellulose and hemicellulose were hydrolyzed to reduce their mean size to 198 ± 14.9 nm and 141.2 ± 23.7 nm respectively. SEM-EDX and TEM analysis confirmed that intense ultrasonication followed during the process resulted in the formation of spherical nanocellulose and nanohemicellulose. XRD analysis revealed that the isolated nanocellulose has a crystallinity of 88.66% and the nanohemicellulose were found to be semicrystalline. Spherical nanoparticle reinforced films exhibited uniformity and enhanced polymeric structure. Films prepared using 0.75% nanocellulose showed a higher tensile strength of 5.22 ± 0.05 MPa, Young’s modulus of 200.83 ± 6.94 MPa and films reinforced with 0.75% nanohemicellulose resulted in tensile strength of 6.16 ± 0.03 MPa and Young’s modulus of 356.88 ± 8.01 MPa respectively. The Water Vapor Permeability significantly reduced to 0.31 ± 0.08 × 10−9 gm−1 s-1 Pa-1 on addition of 1% nanocellulose and 0.32 ± 0.05 × 10-9 gm-1 s-1 Pa-1 on addition of 1% nanohemicellulose respectively. Onset degradation temperature increased to 290 °C and 280 °C for nanocellulose and nanohemicellulose loaded films respectively. Results show that high purity hemicellulose was also attained along with cellulose by the proposed method and intense ultrasonication coupled with acid hydrolysis results in the production of spherical nanoparticles. Results also reveal that the underutilized hemicellulose poses an excellent prospect in film fabrication when reinforced as a nanoparticle by improving the mechanical and water vapor permeability.