Lateral flow nucleic acid biosensors (LFNABs) have attracted extensive attention due to their rapid turnaround time, low cost, and results that are visible to the naked eye. One of the key steps to develop LFNABs is to prepare DNA–gold nanoparticle (DNA–AuNP) conjugates, which affect the sensitivity of LFNABs significantly. To date, various conjugation methods—including the salt-aging method, microwave-assisted dry heating method, freeze–thaw method, low-pH method, and butanol dehydration method—have been reported to prepare DNA–AuNP conjugates. In this study, we conducted a comparative analysis of the analytical performances of LFNABs prepared with the above five conjugation methods, and we found that the butanol dehydration method gave the lowest detection limit. After systematic optimization, the LFNAB prepared with the butanol dehydration method had a detection limit of 5 pM for single-strand DNA, which is 100 times lower than that of the salt-aging method. The as-prepared LFNAB was applied to detect miRNA-21 in human serum, with satisfactory results. The butanol dehydration method thus offers a rapid conjugation approach to prepare DNA–AuNP conjugates for LFNABs, and it can also be extended to other types of DNA biosensors and biomedical applications.