The aim of the present work was to investigate the preparation of low molecular weight heparin (LMWH) nanoparticles (NP) as potential oral heparin carriers. The NP were formulated using an ultrasound probe by water-in-oil-in-water (w/o/w) emulsification and solvent evaporation with two biodegradable polymers [poly-epsilon-caprolactone, PCL and poly(D,L-lactic-co-glycolic acid) 50/50, PLGA] and two non-biodegradable positively charged polymers (Eudragit RS and RL) used alone or in combination. The mean diameter of LMWH-loaded NP ranged from 240 to 490 nm and was dependent on the reduced viscosity of the polymeric organic solution. The surface potential of LMWH NP prepared with Eudragit polymers used alone or blended with PCL and PLGA was changed dramatically from strong positive values obtained with unloaded NP to negative values. The highest encapsulation efficiencies were observed when Eudragit polymers took part in the composition of the polymeric matrix, compared with PCL and PLGA NP exhibiting low LMWH entrapment. The in vitro LMWH release in phosphate buffer from all formulations ranged from 10 to 25% and was more important (two- to threefold) when esterase was added into the dissolution medium. The in vitro biological activity of released LMWH, determined by the anti-factor Xa activity with a chromogenic substrate, was preserved after the encapsulation process, making these NP good candidates for oral administration.