This paper describes the fabrication of a nanoslit membrane-integrated fluidic chip (Nanoslit-Chip) used for trapping and concentrating micro-/nano-particles of desired size and its application in detecting biological molecules based on target-induced particle aggregation. To trap particles of a specific size, a large scale uniform sized nanoslit fluid channel array is fabricated on a silicon dioxide membrane. A small number of fluorescence labeled particles in a large volume of solution are concentrated into a monolayer of particles in a small nanoslit membrane, which enables us to effectively quantify them via fluorescence intensity. In addition, the particles of desired size (1.8 μm) are readily separated from the mixture of particles with a different size (450 nm) in Nanoslit-Chip size, and then quantified via fluorescence measurements. Finally, the Nanoslit-Chip is successfully applied to the sensitive detection of proteins by target-induced particle aggregation, trapping, and quantification. This shows its potential as a biological and clinical device for quantitative and sensitive detection of biological molecules.