The pre-analytical phase of biological analyses is critical as it registers the most errors encountered within the testing process. This study aimed at assessing the knowledge, attitudes and practices of healthcare workers on the pre-analytical phase in haematological laboratory. A cross-sectional study using an auto-administered questionnaire and direct observations of haematological analyses’ prescribers, blood samples and laboratory staff were conducted from June to August 2020. The questionnaires were designed to gather the socio-professional characteristics, knowledge and attitudes of participants. The observation grids were focused on their practices in haematological analyses requesting, blood samples drawing, transportation and reception at the laboratory. A total of 388 respondents were included. Their average seniority in the profession were 11.4 ± 3.8 years and 13.6% received refreshing training in the last three years. All the laboratory agents, 94.1% of analyses’ prescribers and 76% of blood samplers had good knowledge and attitudes. Moreover, 83% of the request forms, 29.2% of blood samples collection, transportation and 74.8% of blood specimens’ reception at the laboratory were rated good. This study reported some inadequate knowledge, attitudes and practices in the pre-analytical phase that call for reinforcement of basic and continuing trainings and implementation of rigorous technical procedures.