Size-controlled lipid nanoparticle (LNP)-based DNA/RNA delivery is a leading technology for gene therapies. For DNA/RNA delivery, typically, a cationic lipid is used to encapsulate DNA/RNA into LNPs. However, the use of the cationic lipid leads to cytotoxicity. In contrast, noncationic NPs, such as exosomes, are ideal nanocarriers for DNA/RNA delivery. However, the development of a simple one-step method for the production of size-controlled noncationic NPs encapsulating DNA/RNA is still challenging because of the lack of electrostatic interactions between the cationic lipid and negatively charged DNA/RNA. Herein, we report a microfluidic-based one-step method for the production of size-controlled noncationic NPs encapsulating small interfering RNA (siRNA). Our microfluidic device, named iLiNP, enables the efficient encapsulation of siRNA, as well as control over the NP size, by varying the flow conditions. We applied this method to produce size-controlled exosome-like NPs. The siRNA-loaded exosome-like NPs did not show in vitro cytotoxicity at a high siRNA dosage. In addition, we investigated the effect of the size of the exosome-like NPs on the target gene silencing and found that the 40-50 nm-sized NPs suppressed target protein expression at a dose of 20 nM siRNA. The iLiNP-based one-step production method for size-controlled noncationic-NP-encapsulated RNA is a promising method for the production of artificial exosomes and functionally modified exosomes for gene and cell therapies.