The nanoassembly of nearly monodisperse nanoparticles (NPs) as uniform building blocks to engineer zirconia (ZrO2) nanostructures with mesoscopic ordering by using a template as a fastening agent was explored. The mesophase of the materials was investigated through powder X-ray diffraction and TEM analysis (TEM) and N2 sorption studies. The TEM results revealed that the mesopores were created by the arrangement of ZrO2 NPs with sizes of 7.0–9.0 nm and with broad interparticle pores. Moreover, the N2 sorption study confirmed the results. The surface chemical analysis was performed to estimate the distribution of Zr, O, and S in the sulfated ZrO2 matrices. The materials in this study displayed excellent catalytic activity in the biodiesel reaction for effective conversion of long-chain fatty acids to their methyl esters, and the maximum biodiesel yield was approximately 100 %. The excellent heterogeneous catalytic activity could be attributed to the open framework, large surface area, presence of ample acidic sites located at the surface of the matrix, and high structural stability of the materials. The catalysts revealed a negligible loss of activity in the catalytic recycles.