Subcritical water extraction (SWE) is an environment-friendly and energy-efficient technology. However, the coupling of ultrasound and SWE for peptides production is not reported. In this study, we performed a two-step strategy to obtain optimal peptide extraction from Spirulina platensis. Firstly, by improving extraction kettle in the original SWE equipment, the extraction rate was increased from 10.30% to 40.20%; Secondly, by coupling SWE with ultrasound and subsequent process optimization, the extraction rate was elevated to 74% under the process conditions: temperature 153 °C, power 221 W, time 64 min. Notably, the extraction rate was 45.80% using traditional ultrasound + freeze-thaw method, and the longer time (at least 16 h) was required. Moreover, the molecular weight distribution indicated that the traditional ultrasound + freeze-thaw method primarily extracted proteins (>5000 Da), while the newly developed ultrasound + subcritical water method primarily extracted peptides (180–5000 Da). This provides a one-step method to obtain peptides without enzyme hydrolysis. [ABSTRACT FROM AUTHOR]