Gold nanoparticles (AuNPs) display irreversible aggregation on exposure to sub-zero temperatures along with changes in the characteristic absorption peak, size, shape etc. Present study, has investigated partial cryoaggregation of AuNPs, physiochemical characterization and in-vitro interaction of the aggregates with Neuro-2a and Caco-2 cell lines. Additionally, time dependent changes in the optical properties of AuNPs upon freezing, have been investigated for use in time-temperature indicators for freeze sensitive products. AuNPs (20 nm and 40 nm size), were exposed to sub-zero temperatures of − 80 °C and − 196 °C, respectively, and the time duration of freezing was adjusted so as to allow partial/complete volume of the suspension to freeze, resulting in a mixed population of monodispersed and aggregated AuNPs. Partial cryoaggregation of AuNPs was observed to be independent of the size of the nanoparticles, since the change in the characteristic absorption spectra with increase in the time duration of freezing was observed to be same for both the types of AuNPs. Broadly, the aggregation state of the AuNPs did not affect the biocompatibility and no deleterious effect were observed in group of cells treated with partially/completely aggregated AuNPs as compared to cells treated with monodispersed AuNPs. Furthermore, coldness indicators based on the phenomenon of partial cryoaggregation of AuNPs, were sensitive towards estimating minute fluctuations in the freeze history of a product. Present study, is first report on evaluating the phenomenon of progressive cryoaggregation of AuNPs, its physiochemical characterization and in vitro interaction with cells, along with use in coldness indicators for enhanced sensitivity.