This study investigated the efficacy of combining binaural beat music (BBM) with rhythmical photic stimulation at the frequency in alleviating anxiety among daycare staff and explored its impacts on daycare staff with different anxiety levels. A quasi-experimental research design was adopted, which included three interventions: BBM, BBM integrated with rhythmical photic stimulation, and relaxation music (control group). Participants completed a questionnaire prior to the first intervention, which included personal demographic information and the Beck Anxiety Inventory. The effects of these interventions on anxiety relief among daycare staff were evaluated through heart rate variability (HRV), brain waves, and blood pressure before and after the interventions. Statistical analysis primarily employed the Friedman test to analyze the differences in changes in HRV, brain waves, and blood pressure before and after the interventions. A total of 40 individuals participated in this study (16 males and 24 females), with an average age of 31.73 ± 8.83 years. The results showed that, compared to BBM alone, BBM integrated with rhythmical photic stimulation significantly reduced the normalized low/high frequency (nLF/nHF) ratio in participants with moderate anxiety ( < 0.05). The results suggest that BBM integrated with rhythmical photic stimulation may serve as an intervention for the prevention and relief of anxiety by regulating an individual's autonomic nervous system. However, further research is required to confirm these findings.