Gold nanoparticles (AuNPs) are increasingly being used as diagnostic and therapeutic agents owing to their excellent properties; however, there is not much data available on their dynamics in vivo on a single particle basis in a single mouse. Here, we developed a method for the direct analysis of nanoparticles in trace blood samples based on single particle inductively coupled plasma-mass spectrometry (spICP-MS). A flexible, highly configurable, and precisely controlled sample introduction system was designed by assembling an ultralow-volume autosampler (flow rate in the range of 5-5000 μL/min) and a customized cyclonic spray chamber (transfer efficiency up to 99%). Upon systematic optimization, the detection limit of the nanoparticle size (LODsize) of AuNPs in ultrapure water was 19 nm, and the detection limit of the nanoparticle number concentration (LODNP) was 8 × 104 particle/L. Using a retro-orbital blood sampling method and subsequent dilution, the system was successfully applied to track the dynamic changes in size and concentration for AuNPs in the blood of a single mouse, and the recovery for the blood sample was 111.74%. Furthermore, the concentration of AuNPs in mouse blood reached a peak in a short period of time and, then, gradually decreased. This study provides a promising technique for analyzing and monitoring the size and concentration of nanoparticles in ultralow-volume blood samples with low concentrations, making it a powerful tool for analyzing and understanding the fate of nanoparticles in vivo.