The microphase separation and crystallization of all-conjugated poly(3-butylthiophene)-b-poly(3-dodecylthiophene) (P3BDDT) diblock copolymers were systematically investigated by temperature-resolved wide-angle X-ray diffraction (WAXD), Fourier transform infrared (FTIR), and synchrotron small-angle X-ray scattering (SAXS) in a step-by-step cooling process. The WAXD, FTIR, and SAXS results revealed that the crystallization of poly(3-butylthiophene) (P3BT) blocks with shorter alkyl side-chains in high temperature region led to the formation of microphase-separated lamellar structure consisting of P3BT crystalline domains and P3BT/poly(3-dodecylthiophene) (P3DDT) amorphous domains, followed by a rapid crystallization of P3DDT blocks in relatively low temperature region, which occurred in the amorphous domains between P3BT crystalline domains. Analysis of the one-dimensional (1D) correlation function derived from SAXS data suggested that the previously crystallized P3BT segments could confine the size of P3DDT crystalline subdomains and possibly contribute to the rapid increase in the P3DDT crystallinity.