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Acoustic frequency technology can promote the growth of plants in horticulture, but the promotion mechanism is not clear. Therefore, the method of spontaneous acoustic frequency (SAF) measurement was explored, and the spontaneous spectrum of cucumber seedlings was examined. Using a laser PDV-100 vibrometer, the SAF of cucumber seedlings in different locations (main vein, mesophyll and stem) and different environments (light intensity changes and drought stress) was measured in a semi-anechoic room by non-contact measurement. The power spectrum and autocorrelation of SAF were analyzed and the spontaneous spectral characteristics of cucumber seedlings were obtained. The results showed that the basic frequency of cucumber seedlings’ SAF was in 4.98~5.86 Hz and the spontaneous acoustic signal variations under different environments in the three parts of the cucumber were consistent.