The invention discloses a microfluidic atom cavity, an on-chip atomic clock chip and a preparation method. The method includes steps: a silicon substrate with a microfluidic channel and a borosilicate glass assembly wafer with a glass microcavity structure corresponding to the microfluidic channel are bonded to form a closed system, the closed system comprises a glass atom cavity, a reactant partition microfluidic channel and a reactant microcavity, reactant particles of materials necessarily required by atomic clock generation are arranged in the reactant microcavity, the glass atom cavity is communicated with the reactant microcavity through the reactant partition microfluidic channel, the aperture of the reactant microcavity on a bonding surface is smaller than that of the glass atom cavity, the minimum width of the reactant partition microfluidic channel is no larger than the minimum particle diameter of the reactant particles, and the glass micro cavity is provided with a light incidence plane. The smaller microcavity containing the reactant powder is connected with the larger microcavity required by forming of the spherical glass microcavity, forming of a spherical rubidium steam cavity and sealing of gas can be completed at one step, fine sealing performance is achieved, no impurities can be led in, performance of an atomic clock can be improved, the spherical rubidium steam cavity can be integrated into a microchip-level atomic clock system, planar packaging of the atomic clock can be realized, and size of the atomic clock is reduced effectively.