A method to determine postmortem interval (PMI) based on environmental temperature and the concentrations of vitreous humor (VH) molecules were explored. Rabbit carcasses were placed in a chamber at 5, 15, 25, or 35 degrees C, and 80-100 mu L of VH was collected with the double-eye alternating micro-sampling method every 12 h. A Roche DPPI biochemical analyzer was used to measure the concentrations of six substances in VH samples. The interpolation function model and mixed-effect model were employed for data fitting to establish equations for PMI estimation. The concentrations of K+, P, Mg2+, creatinine (CRE), and urea nitrogen (UN) exhibited an upward trend with increasing PMI in all temperature groups, while the concentration of Ca2+ showed a downward trend. Validation results using K+ and Mg2+ ions revealed that the mixed-effect model provided a better estimation than the interpolation function model using the data from our experiment. However, both models were able to estimate PMI using temperature and VH molecule concentrations.