Thiolated functional nucleic acid-gold nanoparticle conjugates (FNA-AuNPs) are the core recognition elements in biosensors. The long-term functional stability (LTFS) is critical for their practical applications and, however, has been overlooked. Here we report on the huge effects of multiple experimental factors on LTFS, including spacer- and buffer-composition, secondary structures of FNAs, and surface blocking. We quantitatively determined these effects by measuring the relative hybridization capacity (RHC, the relative amount of complementary DNA hybridized with the same amount of conjugates) for linear DNA-AuNP or the relative signal change generated by their function (RSC-F) for molecular beacon (MB) and G-quadruplex (G4)-AuNPs. There is a positive relationship between the spacer affinity [oligoadenine (A10) > oligothymine (T10) > oligoethlyene glycol (EG18)] of the linear DNA probes and the LTFS. The LTFS of linear DNA-AuNP in phosphate buffer (PB) was much better than that in Good's buffers such as HEPES, Tris, and MES. The secondary structure of FNAs also strongly impacted the LTFS, showing the substantially decreased LTFS from G4- to linear DNA- to MB-AuNPs, where EG18 spacer was used for all these conjugates. The surface blocking of FNA-AuNPs greatly improved the LTFS. We experimentally determined that the LTFS of FNA-AuNPs was directly related to the dissociation of DNAs caused by the in situ generated H2O2 due to the oxidase activity of AuNP and thereby oxidation of Au-thiol bonds. The oxidase activity of AuNP was favored at high temperature, low pH, high AuNP concentration, high Good's buffer concentration, and high salt concentration, corresponding well with the positive effects of high affinity spacer, PB, and surface blocking on the LTFS of FNA-AuNPs. Our study has implications on both fundamental surface science and practical applications.