High-temperature stability of organic field-effect transistors (OFETs) is critical to ensure its long-term reliable operation under various environmental conditions. The molecular packing of donor-acceptor (D-A) conjugated polymers is closely related to the electrical performance stability in OFETs. Herein, we choose poly[[N,N′-bis(2-octyldodecyl)-naphthalene-1,4,5,8-bis(dicarboximide)-2,6-diyl]-alt-5,5′-(2,2′-bithiophene)] as a modal system to reveal the relationship between the molecular stacking and electrical stability in high-temperature environment. The results demonstrate that the films with D-A moieties in alternate stacking have better electrical thermal stability compared to normal donor-donor (D-D) stacking. The D-A stacking configuration alternates donor and acceptor units along the out-of-plane direction, while the D-D stacking involves D-D and A-A stacking separately. The structural transition from D-D to D-A is captured at a treated temperature range of 225–250°C. Owing to the tighter packing arrangement along the π-π and lamellar directions, the electron mobility of the D-A stacked films reaches up to 0.23 cm2/V·s, a 50% increase as compared to the D-D stacking films. Furthermore, the D-A stacked films indicate superior electrical performance stability with mobility retaining 100% at 250°C during high-temperature cycling tests. This result highlights that the manipulation of conjugated polymer closely stacked structures can significantly enhance the thermal stability and durability of semiconductor devices.