The invention relates to an extraction and separation method of a trace uranium element in a liquid sample and belongs to the technical field of trace analysis sample pretreatment. The method primarily utilizes a mixture of di-iso-amyl pentylphosphonate and trioctylphosphine oxide as an extraction agent, strictly controls a concentration of a nitric acid leaching solution, allows the uranium element to have a higher distribution coefficient and better adsorptive capacity, and therefore, achieves a better extraction and separation effect; and an ammonium carbonate solution with a specific concentration is used as an eluent to allow the uranium element to have a higher recovery rate. The extraction and separation method can achieve concentration of the trace uranium element in the liquid sample simply, conveniently and rapidly and separation of some interference heavy metal ions at the same time, and improves stability and reliability of a determination result of the trace uranium element in the liquid sample.