A novel, enzyme-free and aptamer-based colorimetric platform for protein detection has been developed, which takes advantage of aptamer-functionalized magnetic beads (MBs) for target capture, concentration and separation, and aptamer-conjugated gold nanoparticle (AuNP)-catalyzed color bleaching reaction of methyl orange (MO) to generate the colorimetric signals. It was demonstrated that the proposed colorimetric sensing strategy enables simple, cost-effective, sensitive and specific thrombin detection without the use of any enhancing solutions and enzymes. Herein, by naked eye observation, we can detect the human thrombin with a detection limit of approximately 320 pM, which can be further decreased to 30 pM with the help of a UV-vis instrument. In addition, this method also works for targets with two or more binding sites.