In recent years, disposal of used cooking oil has drawn everyone attention due to its detrimental and hazardous effects upon health and environment. However, used cooking oil has great potential to be a fuel supplement after its appropriate processing for biofuel formulation, globally. Attributed to the various advantages of microemulsion-based hybrid biofuel (MHBF) technique, the present experimental investigation has focused to investigate the effects of used cooking oil (UCO)-based hybrid microemulsion biofuel (UCOMHBF), added with 2-ethylhexyl nitrate (2-EHN) (cetane enhancer) upon performance, combustion and emission characteristics of a 4-stroke single-cylinder CI engine. 2-EHN was added in UCOMHBF (UCO (55%) (vol.%), anhydrous ethanol (E) (28%) (vol.%) and 2-butanol (B) (17%)) in proportion of 500 (UCOMHBF500), 1000 (UCOMHBF1000) and 1500 (UCOMHBF1500) ppm, respectively. The brake-specific fuel consumption (BSFC) of CI engine test rig for UCOMHBF1000 was found 8% lower than UCOMHBF and B100 and 2.5% higher for UCOMHBF1500, respectively. Combustion delay was observed with MHBFs w.r.t petrodiesel, but improved combustion was observed with up to 1000 ppm addition of 2-EHN in UCOMHBF at full load condition. Addition of 1500 ppm 2-EHN in UCOMHBF showed uncontrolled flame propagation at the end of the compression stroke which resulted in incomplete combustion during power stroke. 2-EHN addition in UCOMHBF has also lowered the mean of nitrogen (NO x ) in the range of 2.06-33.33% w.r.t petrodiesel. Hence, it can be concluded that UCOMHBF1000 has shown the superior characteristics from all other UCOMHBFs.