This study developed a complexometric titration simulation program based on LabVIEW. With built-in approximation and rigorous algorithms, the program can easily simulate and plot a single or a group of complexometric titration curves. On the basis of this program, we developed and implemented an online course to guide undergraduates to (i) compare the differences between the two titration types EDTA (Y) to Metal (M) and M to Y, (ii) explore the influencing factors of a titration jump, (iii) explore the conditions for an accurate titration, and (iv) find evidence for the existence of complexometric equilibrium. Questionnaires from 25 students were used for evaluation. The results showed that the program had high usability, practicability, effectiveness, and user behavior intention. Besides, 92% of the students were willing to use it more in the future and 11 students completed the test with an average score of 124.6 (full score of 150), and the average score of the modules corresponding to each learning goal was high, indicating that the students had achieved satisfactory learning outcomes.