The present work investigates the effect of using TiO2 Nanoparticles at a different proportion of Azolla algae methyl ester, in a four-stroke single cylinder diesel engine. Biodiesel can be obtained from Azolla algae due to the presence of high oil content. The oil was extracted from Azolla algae by means of the Soxhlet extraction method which was compared to be more effective than the oil obtained by means of hydraulic pressing machine. The obtained oil was converted to biodiesel by means of transesterification process. The fuel properties of the prepared Azolla methyl ester found to confirm with the ASTM standards. TiO2 Nanoparticles were synthesized by means of ball milling process and characterized by means of Scanning Electron Microscope (SEM), Atomic Force Microscopy (AFM), Fourier Transform Infrared (FTIR) and X-Ray Diffraction (XRD) techniques. In the present study, the performance and emission characteristics of diesel engine were analyzed with the addition of TiO2 Nanoparticles to Azolla oil methyl ester (25,50,75 and100 ppm) and compared with that of diesel. The % decrease in Brake Specific Fuel Consumption (BSFC) for B20 + 25, B20 + 50, B20 + 75 and B20 + 100 were found to be 7.81, 12.05, 19.71, and 23.53, respectively, when compared to B20. The % increase in Brake Thermal Efficiency (BTE) for B20 + 25, B20 + 50, B20 + 75 and B20 + 100 were found to be 2.60, 8.49, 8.79 and 13.38, respectively, when compared to B20. The % decrease in Carbon monoxide (CO) for B20 + 25, B20 + 50, B20 + 75 and B20 + 100 were found to be 21.15, 51.92, 54.80, and 57.30, respectively, when compared to B20. The % decrease in Hydro Carbon (HC) for B20 + 25, B20 + 50, B20 + 75 and B20 + 100 were found to be 7.53,8.36,10.04 and 12.97, respectively, when compared to B20. The % increase in Oxides of Nitrogen (NOx) for B20 + 25, B20 + 50, B20 + 75 and B20 + 100 were found to be 5.44, 7.85, 8.67, and 9.16, respectively, when compared to B20. The % decrease in smoke for B20 + 25, B20 + 50, B20 + 75 and B20 + 100 were found to be 18.02, 30.18, 31.98 and 36.04, respectively, when compared to B20. Addition of TiO2 Nanoparticles with the Azolla biodiesel is found to be an effective approach to improve the performance and emission characteristics of a diesel engine without any modification.