The effects of storage conditions and cooking methods on chlorophyll, glucosinolate (GSL), and sulforaphane content in broccoli florets were investigated in this study. For the storage experiment, fresh broccoli florets were stored for 2, 4, and 6 days at 10, 4, and 0 °C with or without 1-methylcyclopropene (1-MCP) treatment. For the cooking experiment, fresh broccoli florets were cooked for 1, 3, 5, 7, and 9 min under three cooking methods, namely, steaming, microwaving, and boiling. Results showed that the contents of chlorophyll, aliphatic GSL, indole GSL, and sulforaphane in broccoli florets of two cultivars decreased with prolonged storage time. The retained contents of chlorophyll, GSLs, and sulforaphane under 0 °C storage condition were significantly higher than those under 10 °C storage condition after 6 days of storage. The sulforaphane content was increased by 1-MCP treatment but differed among varieties. The cooking experiment showed that aliphatic GSL content decreased with increased cooking time under three cooking methods, and indole GSL and sulforaphane contents had a fluctuating trend with increasing cooking time after steaming and microwaving. Sulforaphane content increased by 17.15–50.16% relative to that in fresh broccoli florets and was considerably affected by cooking time. The highest level of sulforaphane content was retained for 7 min during steaming or 5 min during microwaving. Therefore, the combination of 1-MCP treatment and 0 °C storage condition had the best performance in preserving chlorophyll, GSLs, and sulforaphane. Moreover, steaming for 7 min or microwaving for 5 min is a more effective method for preserving the quality and increasing the sulforaphane content of broccoli florets than boiling.