Fluorescence loss spectrum for detecting cold Rydberg atoms with high sensitivity has been obtained based on lock-in detection of fluorescence of 6P3/2 state when cooling lasers of the magneto-optical trap are modulated. The experiment results show that the signal to noise ratio has been improved by 32.64 dB when the modulation depth (converted to laser frequency) and frequency are optimized to 4 MHz and 6 kHz, respectively. This technique enables us to perform a highly sensitive non-destructive detection of Rydberg atoms.