•E-NPCE method was effective and environmentally friendly.•Five indole alkaloids were extracted from Catharanthus roseus at the same time.•E-NPCE can be applied in pilot-scale.In this paper, a novel and green enzyme-assisted negative pressure cavitation extraction (E-NPCE) of five main indole alkaloids vindoline (VDL), catharanthine (CTR), vincristine (VCR), vinblastine (VLB) and anhydravinblastine (AVLB) from Catharanthus roseus leaves was investigated. The incubation conditions were studied using Box–Behnken design (BBD) with response surface methodology (RSM), which revealed these as follows: enzyme amount 0.61% (CB&P, w/w of anhydration material), pH 4.73, incubation temperature 35.87 °C and time 8.62 h. Under the optimal conditions, the extraction yields of VDL, CTR, VCR, VLB and AVLB reached 4.940 ± 0.215, 6.283 ± 0.307, 0.049 ± 0.002, 0.066 ± 0.003 and 0.038 ± 0.001 mg/g, which represented an increase of 38.8%, 51.5%, 48.4%, 37.5% and 58.3%, compared to standard NPCE, respectively. Moreover, this extraction method was also applied on pilot-scale extraction and their yields were 4.761 ± 0.227, 6.093 ± 0.294, 0.047 ± 0.002, 0.064 ± 0.003 and 0.039 ± 0.001 mg/g, respectively. Thus, it demonstrated that E-NPCE could be an alternative method for natural compounds extraction and the most important thing is that this method can be applied to industrial produce.