In our present study, we worked on the preparation of oil-in-water nanoemulsion (NE) and conventional emulsion (CE) with EPA and DHA rich fish lipid oil and comparison of their in vivo intestinal absorption in the single-pass perfusion rat model. Oil-in-Water (O/W) NE and CE using fish lipid oil and Tween 20 and Span 80 as emulsifier/ coemulsifier combination have been successfully prepared by high energy emulsification techniques- high speed homogenization and ultrasonication. The cumulative average droplet diameter of the NEs has been found to be ranged between 78.96. nm and 94.20. nm, with PdI ranging from 0.174 to 0.344, whereas the average droplet diameter of the CEs is 1.58. ±. 0.31. μm. The viscosity and refractive index of the emulsions have been determined. In situ absorption efficiency of the emulsions is measured in laboratory acclimatized adult Sprague-Dawley rats. A comparative study of lipid absorption from NE and CE of similar dilution level, in cannulated small intestine of rats, at time gradient, has been done. Subsequent analysis (e.g. percent volume absorption, percent lipid absorption) have shown that the NE has significantly enhanced the absorption of lipids from the emulsion system, in the small intestine of the rats. This finding about absorption of lipid nanoformulations could be useful for designing lipid-based enteral nutrition therapy for the lipid mal-nutrient patients in future. © 2012 Elsevier Ltd.