A newly identified hemin aptamer with a non-G-quadruplex structure exhibits stronger peroxidase activity and selectivity than traditional G-quadruplex/hemin DNAzymes, addressing challenges such as weak hemin binding, low catalytic activity, and poor selectivity. In this study, we optimized ion activation conditions, refined reaction parameters, and developed a spontaneous recombination method via aptamer splitting to enhance DNAzyme activity and enable activity regulation. The aptamer demonstrated superior performance in enzyme-free sensing, polymerase-assisted amplification, and CRISPR/Cas12a systems, achieving higher sensitivity and improved colorimetric thresholds compared to G-quadruplexes. We have also developed a comprehensive operational guide for aptamer/hemin DNAzymes, which is poised to revolutionize colorimetric sensor signal generation elements.