Cross-sectional study was conducted using panel blood films and questioner to assess detection & identification performance of laboratory professionals' and identify factors affecting the performance of malaria microscopic diagnosis. Study participants had 91.7% (95% CI: 89.96 -93.44) agreement for detection of malaria parasites, 67.63% (95% CI: 64.91 -70.35) species identification agreement for Plasmodium falciparum, 5.08% false positive and 21.04% false negative results. Correct species identification percentage for Plasmodium falciparum were 60.9% (510), Plasmodium vivax 59.17% (371) and Mixed (Plasmodium falciparum and Plasmodium vivax) 25% (53) were also identified in the study. In addition, sensitivity 94.69% (95% CI: 93.02 -96.36) and specificity of 79.71 (95% CI: 75.22 -84.2) were calculated from panel blood film results. The most frequent type of misdiagnosis was 85(40.09%) mixed BFs diagnosed as Plasmodium vivax, 67 (31.6%) mixed BFs as Plasmodium falciparum and 218(26%) Plasmodium falciparum BFs as Plasmodium vivax. Moreover, only 18(8.5%) laboratory professionals were participated in external quality assessment. From multiple logistic regression analysis training was the major factor for species identification percent agreement performance improvement of laboratory professionals. It showed statistical significance with p-value < 0.05 and untrained laboratory professionals were 64% less likely to perform ≥ 85% agreement of species identification. Training of laboratory professionals on malaria microscopic diagnosis help to improve the accuracy and reliability of reported results. This will help to provide the right and recommended medication and patient management.