When the core size of a holey microstructural fiber is close to the wavelength, the light propagation in the core is greatly enhanced and a strong evanescent field is produced around the core. A new air suspended core microstructural fiber with the core diameter of only 2 μm is reported. The air suspended core fiber fabricated by sheet-stacking method has large evanescent field and the unit structure with micrometer-size holes. The air suspended core fiber, whose loss is 0.16 dB/cm at the wavelength of 532 nm, is very suitable for sensing detection of biochemical substances. Using the air suspended core fiber as sensing probe, we establish a simple fluorescence detection system combining with laser technology, and detect the fluorescence properties of bioluminescent labeling material CdTe/CdS/ZnS quantum dots with this system. The limit of the fluorescence quantum dots detected by this system is about 1 nmol/L (corresponds to 3.78×107 quantum dots), which indicates that this system has the properties of high sensitivity and rapid detection speed. The fluorescence detection system based on the air suspended core microstructural fiber provides a new method and idea for the sensitive detection of biomaterials marked by quantum dots.