The development of efficient and selective therapeutic gene delivery methods is a potential medical treatment for intractable diseases. Dry powder inhalers (DPIs) can efficiently deliver drugs locally to the lung. Many reports discuss preparation methods for DPIs. Spray-freeze drying is a method by which highly porous particulates can be prepared. However, altered physical properties after preparation may result in changes in gene expression. In this study, bovine serum albumin (BSA) was added as a lyoprotectant, and 1,2-dioleoyl-3-trimethylammonium propane/cholesterol liposomes/pCMV-Luc DPIs (lipoplex DPIs) were prepared by spray-freeze drying. The mean particle sizes of the lipoplex DPIs prepared without BSA increased by approximately 6.7-fold compared with that of the lipoplexes solution. In contrast, the mean particle sizes of the lipoplex/BSA DPIs increased only slightly. Gene expression was evaluated after the intratracheal administration of the lipoplexes solution, with maximum gene expression observed at 12 h after the administration. In contrast, maximum gene expression of the lipoplex/BSA DPIs occurred at 6h after administration. The gene expression associated with the lipoplex DPIs was significantly lower compared with that of the lipoplex/BSA DPIs at 6 (p<0.01), 12 (p<0.01), and 24h (p<0.05). These variances may be due to the difference in mean particle size between the DPI formulations. The results suggest that BSA is a useful lyoprotectant for dry powder formulation preparations of DNA using the spray-freeze drying method, given that the preparation results in minimal variation of physical properties and gene expression.