In vitro detection of biomarkers requires ultrahigh sensitivity and accuracy. The use of a dual-signal (electrochemical and fluorescent) strategy has the ability of self-calibration and can overcome interferences seen from experimental and environmental factors influencing the experimental results, showing significant advantages in the detection of biomarkers. Here, we propose an enzyme-free and label-free dual-signal aptasensor based on functionalized Apt@antimonene quantum dot (Sb)@ methylene blue (MB)@ZIF-67/Apt@Sb@3,3′,5,5′-tetramethylbenzidine (TMB) @ZIF-67 material to simultaneously detect multiple tumour biomarkers -oestrogen receptor (ER) and human epidermal growth factor receptor-2 (HER2). The aptamer chain of HER2 and ER is adsorbed to cover the porous structure of ZIF-67 through Sb, which has the characteristics of single-chain adsorption, in which the target protein is desorbed later, further encapsulating its internal MB and TMB with good dual-signal properties. After Apt@Sb@MB@ZIF-67/Apt@Sb@TMB@ZIF-67 targeted HER2 and ER biomarkers, the aptamer chains on the Sb quantum dots were desorbed and transferred into the solution, and the MB and TMB signals were released. Negative pressure is applied to the ITO electrode to adsorb and enrich positively charged MB and TMB organic molecules for electrochemical detection, and the supernatant after centrifugation can be directly subjected to fluorescence detection. Therefore, accurate electrochemical and fluorescent dual-signal detection of HER2 and ER from tissue to serum is achieved. Notably, sensitive detection of HER2-ER biomarkers was achieved in only 30 min, with a detection range of 0–10 pg/mL and detection limits of 3.4 fg/mL and 6.9 fg/mL, respectively.