The affinity of aptamers relies on their adaptive folding, but the excessive flexibility of the aptamer backbone usually hampers the folding process. Thus, there is an urgent need to engineer aptamers with more stable and defined structures. Herein, we report a postselection strategy for stabilizing aptamer structures, by fixing both termini of the aptamer with a length-optimized triple helix structure. An anti-lysozyme aptamer was engineered in this way, and its affinity was enhanced by almost 10-fold. An electrochemical aptasensor was designed based on this engineered aptamer, assisted by a DNA tetrahedron as a spacer to orient the aptamer. The aptasensor achieved a 180-fold lower limit of detection than that achieved by the aptasensor without termini-fixed aptamer and exhibited high sensitivity and selectivity toward lysozyme in real red wine samples. This work sheds light on engineering aptamers to achieve enhanced affinity and on the application of aptasensors in complex matrices.