Accidental oil leaks and spills often cause server soil pollution, and in situ remediation is a powerful and economical treatment technology. While during in situ remediation process, unpredicted migration of petroleum hydrocarbon in heterogeneous soil will lead to a long-term source of persistent aquifer contamination. To reduce the migration of petroleum hydrocarbon and effectively improve the in situ remediation efficiency, herein, fungal biomineralization strategy was proposed for the immobilization of petroleum contaminants. A ureolytic fungi strain with crude oil-degradation ability was screened and identified as Chaetomium globosum. When incubated in medium containing Ca²⁺ and crude oil, a mineral corona with spiny nanoparticles was formed at the edge of oil and the interface characters were analyzed using fluorescent pH and dissolved oxygen (DO) sensing films, respectively. Results indicated that biominerals preferred to aggregate around the edge of crude oil, providing favorable microenvironment for fungal growth and then leading to the increase of pH in the microenvironment, eventually accompanied by the formation of mineral corona. The mineral corona with numerous nanoparticles may act as a solid and stable shell, limiting or reducing the mobility of crude oil, and providing enough time for fungal biodegradation. After 28 days incubation, oil-contaminated soil treated with fungal biomineralization showed better immobilization ability for total petroleum hydrocarbon (TPH) under simulated acid-rain condition and higher TPH removal efficiency. This is the first demonstration for the immobilization of oil through fungal biomineralized nanoparticles, thus providing a novel strategy for the in situ remediation of oil-contaminated sites.