Pemetrexed (PEM) is used for treatment of non-small cell lung cancer. However, PEM has disadvantages like fast elimination, low bioavailability, poor tumor cell selectivity, and penetration. Thus, there is a need for using pemetrexed delivery system to increase the anticancer effect of drug in lung cancer cells and to minimize its side effects. The purpose of this study is development of alginate/chitosan nanoparticles (ACNP) that have biodegradable and non-toxic structure for effective delivery of PEM for lung cancer therapy. In the present study, ACNP were prepared using the ionic gelation method, and pemetrexed was loaded via the adsorption method. Drug adsorption efficiency was calculated to be 57.80% and characterization studies were performed. In vitro drug release tests were carried out at pH levels of 5.5 and 7.4 with pemetrexed-loaded alginate/chitosan nanoparticles (PACNP) and free pemetrexed, and both the results were subsequently compared. Up to 11% release yield was observed at pH 5.5, and the yield reached up to 7% in pH 7.4 in the 25 hours. This nanoparticle system could be investigated in vitro and in vivo in further studies for controlled release of pemetrexed.