Carotenoids are known for their lower environmental pollution, lower costs, shorter extraction time compared to regular extraction methods and higher extraction efficiency. Ultrasound has significant advantages in the extraction of a great number of biologically active compounds including carotenoids. Benefits of using ultrasound for extraction of various compounds are numerous and include effective mixing and micro-mixing, more efficient energy transfer, reduced thermal and concentration gradients, reduced temperature. The aim of this work was to investigate the influence of two-frequency ultrasound main parameters such as ultrasonic intensity, sonication time, and temperature. Application of two-frequency ultrasound (simultaneous influence of ultrasound with frequency of 20–46 kHz, intensity of 1.5–2.5 W/cm2 and frequency of 1.0–2.0 MHz, intensity of 2.0–3.0 W/cm2) raises the percentage of β-carotene extraction from biomass of the fungus Blakeslea trispora from 90–92% up to 98–99% in comparison with one frequency ultrasound. The recommended oil temperature for the extraction process is 35°C, because lower temperature leads to reduction of percentage of β-carotene extraction, and higher temperature promotes unjustified increase of expenditure of energy