We chose a series of isoindigo-based conjugated polymer (IIDDT, IIDDT-C3 and IIDDT-C4) with different length of side chains and bifurcation positions to investigate the relationship between the degree of alignment and the length of side chains and bifurcation positions. We found that the dichroic ratio was increased from 2.37 to 5.23 when the side chain was longer and the bifurcation position was away from the backbone. The π–π stacking distance was decreased from 3.67 Å to 3.61 Å when the bifurcation position was away from the backbone because of its smaller hindrance and the d-spacing of the (1 0 0) was increased from 20.06 Å to 25.21 Å when the side chain was longer. All the polymers were adopted an edge-on orientation with the backbone paralleled with the long axis of fibers. The weak interaction of side-chain in IIDDT-C4 was beneficial for the molecules being rearranged in parallel during the contact line receding and the strong π–π interaction could accelerate the interchain assembly of the parallel molecules through π–π interaction to form aligned fibers.