The sensitive and accurate detection of thrombin (Tb) is of significance for early diagnosis of thrombosis, vascular injuries, and cancers. A dual-signal aptasensor with effective signal amplification is a promising approach to improve accuracy and sensitivity simultaneously for detection of Tb. Herein, an electrochemistry (EC)-colorimetry dual-readout aptasensor was proposed for Tb detection based on a multifunctional probe, MB@ZIF-90/Cu(I)/(Ⅱ). Methylene blue (MB), as an electroactive and colorimetric dual-signal molecule, was encapsulated into ZIF-90 along with introducing Cu species to form MB@ZIF-90/Cu. After post-treatment heating, MB@ZIF-90/Cu(I)/(Ⅱ) formed with the catalysis toward MB even without the addition of substrate. The catalysis was further enhanced by the “confinement effect” of Cu(I)/Cu(Ⅱ) system and MB in the pore for amplified electrochemical signals. While, the unbounded probes were collected for colorimetric analysis. As a result, EC-colorimetric dual-readout aptasensor exhibits wider linear ranges of 1 pM–10 μM and 0.1 nM–100 μM and lower detection limit down to 0.3 pM and 0.03 nM for Tb, respectively. The mixed-valence MOFs-based dual-signal aptasensor achieved ultrasensitive detection of Tb with more reliable results, revealed its promising application in clinical diagnosis of Tb and even other biomarkers.