In response to the issues of poor water flooding efficiency, low oil production rates, and low recovery rates during the high-water-cut period in the low-permeability reservoirs of the Mutou Oilfield, the non-steady-state (NSS) CO2 huff-n-puff oil recovery technology was explored. The NSS CO2 huff-n-puff can improve the development effect of low-permeability reservoirs by replenishing the reservoir energy and significantly increasing the crude oil mobility. Experimental investigations were carried out, including a crude oil and CO2–crude oil swelling experiment, minimum miscibility pressure testing experiment, high-temperature and high-pressure microfluidic experiment, and NSS CO2 huff-n-puff oil recovery on-site pilot test. The experimental results showed that the main mechanisms of NSS CO2 huff-n-puff include dissolution, expansion, viscosity reduction, and swept volume enlargement, which can effectively mobilize the remaining oil from the various pore throats within the reservoir. The high-temperature and high-pressure microfluidic experiment achieved an ultimate recovery rate of 83.1% for NSS CO2 huff-n-puff, which was 7.9% higher than the rate of 75.2% obtained for steady injection. This method can effectively utilize the remaining oil in the corners and edges, enlarge the swept volume, and increase the recovery rate. Field trials of NSS CO2 huff-n-puff in a low-permeability reservoir in the Mutou Oilfield indicated that it cumulatively increased the oil production by 1134.5 tons. The achieved results and insights were systematically analyzed and could provide key technical support for the application of NSS CO2 huff-n-puff technology in low-permeability reservoirs, promoting the innovative development of this technology.