Oligonucleotides (ONs) have shown great promise as therapeutic agents for various diseases. It is necessary to provide a protocol for preparation of ON-loaded lipid nanoparticles (LNPs) in a reproducible manner on a laboratory scale.A 3-inlet microfluidic (MF) chip-based device was used to synthesize LNPs at the lipid/ON ratio of 10/1 (w/w) and at flow rates ranging from 50 to 1100 μl/min. A series of LNPs containing either antisense oligodeoxyribonucleotide (AS-ODN) or small-interfering RNA (siRNA) were synthesized. Bulk mixing was used as control.The MF method was shown to be particularly useful for synthesis of LNPs loaded with AS-ODN. The optimal range of flow rates for AS-ODN LNPs was found to be 100 to 200 μl/min. MF synthesis produced LNPs with lower polydispersity values. However, the MF was less effective in preparing LNPs loaded with siRNA, which may have been due to greater rigidity of double-stranded siRNA comparing to single-stranded AS-ODN.MF technology is a simple, affordable and reproducible method for production of ON-LNPs.