A novel method was developed for the one-pot synthesis of ultrafine poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs), using an emulsion solvent evaporation formulation method. Using either cetyltrimethylammonium bromide (CTAB) or poly(ethylene glycol)-distearyl phosphoethanolamine (PEGPE) as an oily emulsifier during the emulsion process, produced PLGA particle sizes of less than 50 nm, constituting a breakthrough in emulsion formulation methods. The yield of ultrafine PLGA NPs increased with PEGPE/PLGA ratio, reaching a plateau at around 85%, when the PEGPE/PLGA ratio reached 3:1. The PEGPE-PLGA NPs exhibited high drug loading content, reduced burst release, good serum stability, and enhanced cell uptake rate compared with traditional PLGA NPs. Sub-50 nm diameter PEG-coated ultrafine PLGA NPs show great potential for in vivo drug delivery systems.