Selection of aptamers with high affinity and good specificity requires multiple rounds of alternating steps of separation and PCR amplification. Herein, we proposed a novel high-efficiency aptamers picking strategy: One-round pressure controllable selection (OPCS). OPCS integrates four types of screening superiority, high-efficiency separation, one-round selection and PCR amplification, synchronous negative selection and targets competition. The controllable screening pressure can be achieved through two approaches, balanced competition by the regulation of protein concentration, and dominant competition by introducing a predatory protein with high concentration. In OPCS process, two proteins were co-incubated with one ssDNA library, and each protein bound its favorable sequences specifically and formed protein-ssDNA complex respectively. Meanwhile, one protein could supply/suffer the picking pressure of affinity and specificity to/from another, which eliminated weakly bound or unbound sequences for each other. Two complexes could be separated and collected conveniently, and aptamers for two proteins obtained synchronously with high affinity and good specificity. This strategy not only provides a more effective way for aptamers selection, but shows great potential in other ligands or drugs selection.