The young ovary wall of Cucurbitaceae plants, including cucumber, melon, watermelon and West Indian cucumber, were used as the material for chromosome preparation. Several main factors including sample size of ovary material, 8hydroxyquinoline pretreatment time and enzymatic hydrolysis time were investigated to analyze their effects to chromosome preparation results, and we also explore how to optimize this method. Using this method, ploidy identification and fluorescence in situ hybridization experiments were carried out on the ovary wall of candidate haploid plants of cucumber. Results show that: (1) the best pretreatment time of young ovary wall is 1 h 30 min for cucumber, 1 h for melon, 55 min for watermelon, 45 min for West India cucumber. To observe clear mitotic metaphase with this optimized method, we should use melon ovary with a length range of 0.2-1 cm, cut the ovary wall material into small pieces with a side length of 1-1.5 mm. And the suitable time for enzyme digestion is 1 h 10 min to 1 h 20 min. (2) The identification results by this method showed that the cucurbitaceae plants cucumber, melon, watermelon and West Indian cucumber had 14, 24, 22 and 24 chromosomes, respectively. The somatic cell chromosome number in the plant identified as haploid was 7. (3) Fluorescence in situ hybridization showed that there were 3 pairs of bright 45S rDNA hybridization signals and 1 pair of 5S rDNA hybridization signals in cucumber chromosomes, and the number of corresponding signals in haploid cucumbers was 7. There were 2 pairs of 45S rDNA and 1 pair 5S rDNA signals in melon, watermelon and West Indian cucumber. Our studies indicated that the ovary wall chromosome preparation procedure is suitable for all these Cucurbitaceae species. Not only can they obtain good mitotic metaphase, but also have the advantages of easy to get material and high chromosome preparation efficiency. Therefore, when it is difficult to obtain root tips of the rare material, the ovary wall chromosome preparation method is an effective method for studying the chromosome number of plants and identifying ploidy. This method is also suitable for fluorescence in situ hybridization.