This textbook is written based on the authors' research works that have been published in various journals along with the lecture notes used when teaching bioinformatics courses at the University of Illinois at Chicago and at Tsinghua University. The content may be divided into two parts. The first part includes three chapters, introducing some basic concepts. Chapter 1 provides biological background in molecular biology for mathematicians. Chapter 2 describes biological databases that are commonly used. Chapter 3 is concerned with alignment methods including global/local alignment, heuristic alignment, and multiple alignment. The second part consisting of five chapters, describes several bioinformatics principles using a rigorous mathematical formulation. Chapter 4 introduces the time-frequency spectral principle and its applications in bioinformatics. In Chapters 5 and 6, two strategies are used, the graphical representation and the natural vector method, to represent biological sequences, and conduct sequence comparison and phylogenetic analysis without alignment. Chapter 7 presents the convex hull principle and shows how it can be used to mathematically determine whether a certain amino acid sequence can be a protein. The last chapter summarizes additional mathematical ideas relating to sequence comparisons, such as new feature vectors and metrics. This part focuses on the governing principle in biology and provides plenty of alignment-free methods, which cannot be found in any other book.