Chromium (Cr) pollution in soil has the greatest threat to human beings as it has a potential to enter into the food chain through contaminated soil. Chromium has no defined role in plant biology and has detrimental effects on plants. Biochar used as soil amendment has been demonstrated to diminish the bioavailability of trace elements. The current study investigated the effect of poultry litter biochar (PLB) on Cr bioavailability and its accumulation in spinach grown on Cr-contaminated soil. Data showed significant decline in bioavailable Cr concentration (DTPA-extraction) with an increasing biochar concentration, especially at 5% application rate in soil. With the 3% and 5% application of PLB, Cr concentration in shoots significantly (P < 0.05) decreased by 32% and 49%, respectively, over the control. The results showed that PLB significantly enhanced the plant growth and biomass over the control. Under Cr stress, the PLB addition increased the catalase and superoxide dismutase activities while decreased malondialdehyde and hydrogen peroxide concentrations as compared to control. Overall, it was concluded that PLB application might be effective in reducing the toxic effects of Cr in spinach and enhancing plant growth.