The subcritical dimethyl ether extraction (SDEE) was applied in the preparation of liver protein powder (LPD) in this paper. As expected, the lipids and water in tuna liver were co-removed by SDEE, and LPD with a high protein content could be obtained. Besides that, the maximal protein content (88.51%) of LPD was obtained by optimized extraction conditions (liquid-solid ratio 34 mL/g, time 70 min and stirring speed 945 rpm). Furthermore, the traditional preparation technique, pH-shift, was used for comparisons. Compared with pH-shift, the higher quality LPD could by obtain by SDEE with slight fishy and excellent yield, because of SDEE could prevent the loss of nutrition constituents and remove more flavor-active components. Moreover, the functional properties of prepared LPDs were compared with soybean protein powder (SPD) and egg white powder (EWP). The results showed that the LPD prepared by SDEE possessed the greater capacities in foaming, foam stability and fat absorption. Therefore, SDEE, with the advantages of no freeze-drying procedure and relatively low pressure, would be a better alternative to prepare high-quality protein powder in the food industry.