The goal of biological samples' cryofixation is to trap a metabolic state as it exists in vivo by rapidly stopping internal reactions. However, obtaining perfect quality of cryofixation for large and high hypermetabolism organ/tissue (such as brain, heart) remains a challenge. The aim of this study was to develop and display a comprehensive and direct method to evaluate cryofixation's process and quality. Here, we adopt a delicate combination of homemade cryo-imaging system with a rat cardiac arrest model that can control cryofixation time optionally. we successfully evaluate the cryofixation time-related nicotinamide adenine dinucleotide (NADH) fluorescence pattern of several coronal sections in rat's brain that suffered from directional funnel cryofixation procedure. Through quantitative analysis of the distribution map of NADH fluorescence, we could obtain a relationship between cryofixation time and well cryofixation volume and then could deduce the cryofixation rates and quality at different time points. Our results also demonstrated that dissection of the temporal muscle of rat could significantly optimize the classical direct funnel cryofixation protocol.