The diffracted X-ray tracking (DXT) method has been applied for capturing conformational changes of single KcsA potassium channels, and which is recording position of X-ray diffraction spot from a gold nanocrystal attached to the channel. In the conventional observation-chamber setup, KcsA channels are fixed on a chemically modified glass surface and they are covered by PDMS. Although this setup enabled to trace a diffraction spot from the gold nanocrystal through a CCD camera, relatively high background noise resulting from X-ray scattering degrades a spatial resolution. This paper report a low-noise microchamber fabricated by three-dimensional UV lithography and an evaluation of its background noise. The proposed micro-chamber consists of two parts; SiN membrane window supported by Si and embedded microchannel in negative photoresist. The combination of SiN membrane window and thin photoresist membrane for micro-chamber ceiling indicated the lowest noise.