A magnetic biochar nanomaterial derived from fungal hyphae was introduced into the sample preparation field. The magnetic fungal hyphae-derived biomass carbon (MFHBC) could be produced by a controllable hydrothermal method. In order to obtain the best sorbent for magnetic solid-phase extraction (MSPE), the reaction conditions containing temperature, time and the consumption of fungal hyphae were investigated. A series of MFHBC materials were characterized by vibrating sample magnetometers, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and transmission electron microscopy. A material with a satisfactory saturation magnetization (21.58 emu g-1) and largest surface area (88.06 m2 g-1) was selected as the sorbent to extract ten typical organochlorine pesticides (OCPs). The extraction conditions were optimized as 20 mL of sample solution with 70 mg of sorbent and 2.0 g of NaCl oscillated at 50 °C for 5.0 min. And the optimum desorption was performed by oscillating sorbent in 1.0 mL acetonitrile for 5.0 min. Then, the MFHBC-based MSPE-GC–MS/MS methods were established for different samples including water samples, tea beverages, and Chinese traditional medicines. The linearities were 10–2500 ng L−1 or 100–25,000 ng kg−1, and the limits of detection were 0.3–13.9 ng L−1 for water sample, 0.1–9.7 ng L−1 for tea beverage samples, 0.1–21.4 ng L−1 for Shenqi Fuzheng injection samples, and 7.2–278.3 ng kg−1 for Astragali Radix decoction pieces. Except for satisfactory repeatability (RSDs ≤13.8%) in intra-day and inter-day tests (n = 3), the reproducibility (RSDs ≤13.5%, n = 3) of MFHBC was acceptable. The methods were applied in the determination of OCPs from above real samples, with the recoveries of 80.5–117.2% and the RSDs (n = 3) <8.9%. The methods were suitable in the sensitive determination of OCPs from simple to complex matrix samples.