The formation of a particle monolayer after the evaporation of a suspension droplet containing micro/nanoparticles is critical for multiple applications, including cell printing, particle immunoassays, and electronics. However, conventional methods for forming monolayers have limitations in terms of the use of solid substrates to fix them. In this paper, a novel method is proposed for forming a microparticle monolayer suspended in air using a through‐hole chip. Utilizing the strong interactions between particles and solid/liquid interfaces at the microscale, a simple yet robust method is developed to fabricate a suspended monolayer via particle suspension evaporation. The particle layers are classified into four types according to the particle concentration and suspension volume, namely, burst, ring, mono and ring, and multi patterns. Reorganization of the particle monolayer is achieved despite the repetitive solvent reinfusion and evaporation processes. Remarkably, the suspended particle monolayer exhibits superior optical characteristics, with a signal‐to‐noise ratio that is 1.78 times higher in fluorescence measurements than the same particles placed on a glass plate. This suspended particle monolayer can be applied in a variety of fields requiring sensitive and reproducible particle detection.