The cucumber (Cucumis sativus L.) exhibits extensive variations in fruit size and shape. Fruit length is an important agronomic and domesticated trait controlled by quantitative trait loci (QTLs). Nonetheless, the underlying molecular and genetic mechanisms that determine cucumber fruit length remain unclear. QTL-seq is an efficient strategy for QTL identification that takes advantage of bulked-segregant analysis (BSA) and next-generation sequencing (NGS). In the present study, we conducted QTL mapping and QTL-seq of cucumber fruit length. QTL mapping identified 8 QTLs for immature and mature fruit length. A major-effect QTL fl3.2, which explained a maximum of 38.87% of the phenotypic variation, was detected. A genome-wide comparison of SNP profiles between two DNA bulks identified 6 QTLs for ovary length. QTLs ovl3.1 and ovl3.2 both had major effects on ovary length with a △ (SNP-index) of 0.80 (P < 0.01) and 0.74 (P < 0.01), respectively. Quantitative RT-PCR of fruit size-related homologous genes localized in the consensus QTL FL3.2 was conducted. Four candidate genes exhibited increased expression levels in long fruit genotypes. Our results demonstrated the power of the QTL-seq method in rapid QTL detection and provided reliable QTL regions for fine mapping of fruit length-related loci and for identifying candidate genes.