This work concentrates on the design and implementation of aptamer-based electrochemical biosensors using a layer-by-layer approach for precise tracking of mucin-1 (MUC1), an important biomarker linked to breast cancer. The electrochemical biosensor was created by modifying a screen-printed carbon electrode (SPCE) with V2C MXene booster and gold nanoparticles (Au-NPs), along with Cd2+ integrated aptamer (AP) (SPCE/V2C-MXene/Au NPs/Cd2+-AP). This biosensor demonstrated high specificity and affinity for MUC1, establishing a sensitive quantification mechanism. The MXene nanolayer was produced and analyzed via TEM, XPS, SEM, AFM, BET, and MAP techniques. It served as a supportive material that enhanced electrochemical conductivity and allowed for the integration of the aptamer (AP) as the biological recognition component. The biosensor was constructed by immobilizing MUC1-specific aptamers onto the surfaces of SPCE/V2C-MXene/Au NPs, enabling selective recognition and binding with MUC1. The recorded signal, corresponding to Cd2+ integrated with AP at SPCE/V2C-MXene/Au NPs/Cd2+-AP, enabled quantitative assessment of MUC1 levels. The findings showed a linear concentration span of 1.0–500 pg/mL for detecting MUC1, achieving a detection limit of 3.45 fg/mL utilizing the SPCE/ V2C-MXene/Au NPs/Cd2+-AP biosensor. The SPCE/V2C-MXene/Au NPs/Cd2+-AP biosensor exhibited a good affinity for the detection of MUC1 in the presence of other breast cancer biomarkers, confirming its selectivity.