The recently reported freezing-based labeling method for constructing DNAAuNP probes is rapid but still requires thiol modification. Here, we evaluated a poly(A)-tagged DNA sequence using the freezing-based labeling method, and the results demonstrated that approximately 10 A bases at the sequence ends are essential. More detailed observations revealed that some DNA sequences tend to form secondary structures and thus shield exposed A bases, resulting in inefficient or failed labeling. However, successful labeling was restored by simply increasing the poly(A)-base number. Building on these discoveries, we developed three kinds of AuNP-based bioprobes, DNA-AuNP, RNA-AuNP, and DNA-enzyme-AuNP, using the freezing-based labeling method. This method was completed in a single mixing step with no need for thiol modification, representing one of the most convenient and lowest cost AuNP bioprobe labeling techniques ever reported. In addition, the resulting AuNP bioprobes were further used to advance CRISPR-based diagnostics through the development of user-friendly colorimetric, fluorescence, and lateral flow detection strategies.