For aptamer selection, the random-region length of an ssDNA library was generally taken in a relatively arbitrary fashion, which may lead to failure for unsuitable target binding. Herein, we coupled high-efficiency capillary electrophoresis (CE)-SELEX and high-throughput sequencing (HTS) to investigate the influences of random-region length. First, one round of selection against programmed cell death-ligand 1 (PD-L1) was performed using ssDNA libraries with random-region lengths of 15, 30, 40, and 60 nt, respectively. A good correlation was observed between candidates' random-region lengths and dissociation constant (Kd), in which the longer sequences presented higher affinity, and the picked Seq 60-1 after one round notably presented a similar affinity toward a reported aptamer through eight rounds. Molecular dynamics (MD) simulation suggested, for PD-L1, the long sequence could supply more noncovalent bonds including hydrogen bonds, electrostatic interactions, and hydrophobic interactions to form a stable protein/aptamer complex. Besides, four other proteins with selective binding performances validated the importance of random-region length. To further investigate how random-region length affects the selection efficiency, a mixed library with random-region lengths ranging from 10 to 50 nt was employed for six rounds of selection against Piezo2. Sequence variations were tracked by HTS, showing the preferential evolution and PCR uncertainty with even higher impact were the main causes. This study suggested random-region length plays a crucial factor, and a mixed library with different random-region sequences can be a worthy choice for increasing the speed of high-affinity aptamer selection. Moreover, the PCR process should be given particular attention in aptamer selection.