Nanoscale morphology is of significance to the electronic properties of semiconducting polymers. Solution-processed poly-3-hexylthiophene (P3HT) has been demonstrated as a promising active-layer material in organic thin film transistors (OTFTs) and solar cells. Controlling the crystallinity of P3HT chains is critical for gaining high-performance devices. Here we demonstrated the immediate crystallization of P3HT induced by two-dimensional MoS2 nanosheets under ultrasonication. The resulting aggregation was attributed to the presence of interaction between the MoS2 nanosheets and P3HT, which could enhance the inter-chain ordering and association of P3HT. The crystallization of P3HT contributed to the 38-fold enhancement in the hole mobility of the thin film as compared to the non-crystallized thin films because of the absence of MoS2. Our approach of using 2D MoS2 nanosheets to induce immediate aggregation of P3HT provides a facile process to control the crystallization of conjugated polymers for the development of high-performance organic electronics.